WO2011048589A2

WO2011048589A2 - Ion exchangers and methods of use thereof

Info

Publication number: WO2011048589A2
Application number: PCT/IL2010/000855
Authority: WO
Inventors: Israel Sekler; Michal Hershfinkel; Yoram Yagil; Raz Palty
Original assignee: Ben Gurion University Of The Negev Research And Development Authority
Priority date: 2009-10-23
Filing date: 2010-10-19
Publication date: 2011-04-28
Also published as: WO2011048589A3

Abstract

This invention provides a method of treating a disease by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. This invention further provides a method of treating a disease by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. This invention also provides compounds and compositions comprising the same for diminishing or abrogating mitochondrial Na⁺/Ca²⁺-Li⁺ exchanger (NCLX) expression or activity, thereby inhibiting apoptosis, or for stimulating or increasing mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression or activity, thereby stimulating or accelerating apoptosis. This invention further provides compounds and compositions comprising the same for modulating apoptosis and treating diseases, disorders and/or conditions related thereto.

Description

ION EXCHANGERS AND METHODS OF USE THEREOF

FIELD OF INVENTION

[001] This invention provides compounds and compositions and uses thereof for the treatment of a disease by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. This invention further provides compounds and compositions and uses thereof for the treatment of disease by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. This invention also provides compounds and compositions comprising the same for diminishing or abrogating mitochondrial Na⁺/Ca²⁺-Li⁺ exchanger (NCLX) expression or activity, for example, for inhibiting apoptosis, or for stimulating or increasing mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression or activity, for example, for stimulating or accelerating apoptosis. This invention further provides compounds and compositions comprising the same for modulating apoptosis and treating diseases, disorders and/or conditions related thereto.

BACKGROUND OF THE INVENTION

[002] Mitochondria play a role in many essential physiological roles in cells, including producing most of the cell's ATP through oxidative metabolism. In addition, mitochondria are known to be potent buffers of cytosolic Ca²⁺. Ca²⁺ is driven into mitochondria through a uniporter which is located in the inner membrane, and export is achieved via multiple mechanisms including a Na⁺/Ca²⁺ exchanger. Calcium flux is believed to modulate the production of ATP and has an important role in modulating Ca²⁺ signaling. Mitochondrial buffering of calcium flux has been noted in many cell types, including pancreatic cells and neurons, and mitochondrial Ca²⁺ has been shown to modulate insulin secretion and the granulosa cells of the adrenal gland.

[003] Another role of mitochondrial Ca²⁺ handling is the control of apoptosis/necrosis. In all metazoans, programmed cell death, or apoptosis, is essential for proper development and maintenance of body homeostasis. Various disease states occur due to aberrant regulation of programmed cell death in an organism. For example, defects that result in a decreased level of apoptosis in a tissue as compared to the normal level required to maintain the steady-state of the tissue can result in an increased number of cells in the tissue. Such a mechanism of increasing cell numbers has been identified in various cancers, where the formation of a tumor occurs not because the cancer cells necessarily are dividing more rapidly than their normal counterparts, but because the cells are not dying at their normal rate. The first gene identified as being involved in a cell death pathway, the bcl-2 gene, was identified in cancer cells and was shown to function by decreasing the likelihood that cells expressing the gene would undergo apoptosis. [004] In comparison to cancer, where the likelihood of a cell undergoing apoptosis is decreased, various pathologies are associated with tissues containing cells undergoing a higher than normal amount of apoptosis. For example, increased levels of apoptosis are observed in various neuropathologies, including Parkinson's disease, multiple sclerosis, Alzheimer's disease, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS). Since nerve cells generally do not divide in adults and new cells are therefore not available to replace the dying cells, nerve cell death occurring in such diseases results in the progressively deteriorating condition of patients suffering from the disease.

SUMMARY OF THE INVENTION [005] This invention provides for the use of an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in the treatment of a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[006] In one embodiment, the mitochondrial Na⁺/Ca²⁺ NCLX exchanger is encoded by a nuclei acid sequence shares at least 95% homology with SEQ ID NOs: 1-3. and in one embodiment, the agent is a microRNA comprising a sequence complementary to a fragment of SEQ NOs: 1-3. In one embodiment, the agent is an antibody specifically recognizing a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said polypeptide has a sequence sharing at least 95% homology with SEQ NOs: 8-10.

[007] This invention provides, in one embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, as well as a vector comprising the nucleic acid.

[008] This invention provides, in one embodiment, an isolated polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said isolated polypeptide has a sequence sharing at least 95% homology with SEQ ID NOs: 8-10.

[009] In one embodiment, this invention provides an isolated nucleic acid comprising a microRNA, which in one embodiment, comprises a sequence complementary to a fragment of SEQ ID NOs: 1-3. which in one embodiment, is a sequence sharing at least 95% homology with SEQ ID NO: 6.

[0010] This invention provides, in one embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding a non-functional mitochondrial Na⁺/Ca²⁺NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO; 13, as well as a vector comprising the nucleic acid. [0011] This invention provides, in one embodiment, an isolated polypeptide comprising an amino acid sequence of a non-functional mitochondrial Na⁺/Ca²⁺NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 14.

[0012] This invention provides, in one embodiment, a method of treating a disease, which is improved by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby treating said disease or in some embodiments, the invention provides for the use of an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of treating a disease, which is improved by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0013] This invention provides, in one embodiment, a method of treating a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na Ca ⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby treating said disease or in some embodiments, the invention provides for the use of an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of treating a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0014] This invention provides, in one embodiment, a method of inhibiting cellular apoptosis comprising the step of contacting a cell with an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby inhibiting apoptosis or in some embodiments, the invention provides for the use of an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of inhibiting cellular apoptosis.

[0015] In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13 or a microRNA having a sequence sharing at least 95% homology with SEQ ID NO: 6, wherein said contacting diminishes or abrogates expression, activity, or a combination thereof, of said exchanger. In another embodiment, the agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0016] This invention provides, in one embodiment, a method of stimulating or accelerating apoptosis comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby stimulating or accelerating apoptosis or in some embodiments, the invention provides for the use of an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of stimulating or accelerating cellular apoptosis.

[0017] In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting up-regulates expression, activity, function, or a combination thereof of said exchanger.

[0018] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition or in some embodiments, the invention provides for the use of an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject.

[0019] In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NOs: 1-3, wherein said contacting diminishes or abrogates expression of said exchanger. In one embodiment, the microRNA shares at least 95% homology with SEQ ID NO: 6. In another embodiment, the agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger. In one embodiment, the pathologic condition is a result of AIDS, or a cardiovascular, neurodegenerative, skeletal, inflammatory or infectious disease or disorder.

[0020] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition or in some embodiments, the invention provides for the use of an agent which v mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject. [0021] In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting stimulates or increases expression of said exchanger. In one embodiment, the pathologic condition is a result of a preneoplastic, neoplastic, autoimmune, or reactive cell disorder.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Cellular and subcellular localization of NCLX.

[0023] Figure 2. Expression of NCLX enhances mitochondrial Ca²⁺ efflux.

[0024] Figure 3. NCLX knockdown by siRNA results in decreased mitochondrial Ca²⁺ efflux.

[0025] Figure 4. Mitochondrial NCLX mediates Na⁺ or Li⁺ dependent Ca²⁺ efflux that is inhibited by siRNA silencing or mutagenesis.

[0026] Figure 5. Measurement over time of systolic blood pressure in hypertensive rats treated with CGP 37157.

[0027] Figure 6. Comparison of systolic blood pressure in control and hypertensive rats treated with CGP 37157.

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention provides novel nucleic acids, compounds and methods for modulating calcium flux and cellular apoptosis, and therapeutic applications, unavailable in the state of the art to date, arising from their utilization in myriad pathologies.

[0029] Mammalian cells respond to external stimuli in part by transient changes in cytoplasmic

2+

Ca concentration, often via interaction with Ca-binding proteins such as calmodulin. Normal cell functions require that cytoplasmic Ca²⁺ concentration be rigorously maintained, and nanomolar changes in Ca²⁺ concentration can initiate major metabolic events. Mitochondria fill several essential physiological roles in mammalian cells, including playing a central role in the regulation of cytoplasmic Ca²⁺ in cells. Their best recognized function is to produce the bulk of the cell's ATP through oxidative metabolism. In addition to this fundamental role as the cell's primary energy supplier, mitochondria are also potent buffers of cytosolic Ca²⁺. The large negative potential across the inner membrane drives Ca²⁺ into mitochondria through a uniporter located in the inner membrane, and export is achieved via multiple mechanisms, including an Na⁺/Ca²⁺ exchanger. Uptake and release of Ca²⁺ by mitochondria has an important effect on Ca²⁺ signaling. Their high rate of uptake enables mitochondria to play a dominant role among several mechanisms (including Na⁺/Ca²⁺ exchange and Ca²⁺-ATPases) for clearing large loads of cytosolic Ca²⁺. By slowly releasing stored Ca²⁺, mitochondria can prolong the period of Ca²⁺ elevation in response to a transient episode of Ca²⁺ influx. These actions may have important effects on such Ca²-dependent processes as exocytosis and synaptic transmission. Thus, mitochondria act like a slow, nonsaturable, nonlinear buffer for intracellular Ca²⁺; they sequester Ca²⁺ during periods of rapid Ca²⁺ entry and release it slowly after Ca²⁺ entry has ceased.

[0030] Mitochondrial Ca²⁺ efflux is fundamental to a wide range of cellular activities, including control of oxidative phosphorylation, modulation of cytosolic calcium signals, cell death and secretion. By catalyzing Na⁺-dependent Ca ⁺ efflux, the putative mitochondrial Na⁺/Ca²⁺ exchanger plays a fundamental role in regulating mitochondrial Ca²⁺ homeostasis. Although it is thought that a Na⁺-dependent mechanism regulates Ca²⁺ efflux from the mitochondria, the Na⁺/Ca²⁺ exchanger, NCLX, was only identified herein as the transporter mediating this process. Using immunoblot, immunofluorescence and immunoelectron microscopy, NCLX was identified on the mitochondrial inner membrane (Figure 1). Functional analysis, employing Ca²⁺ imaging techniques, indicated that mitochondrial Ca²⁺ efflux is enhanced upon heterologous expression of NCLX (Figure 2) and reduced by NCLX silencing with siRNA (Figure 3). In addition, the NCLX-dependent mitochondrial Ca²⁺ transport activity is inhibited by CGP-37157 and exhibits Li⁺-dependent Ca²⁺ transport, both hallmarks of the mitochondrial Na⁺-dependent Ca²⁺ efflux. In addition, mitochondrial Ca²⁺ transport activity, mediated by NCLX, is blocked by a mutation at a catalytic site of NCLX (Figure 4),

[0031] This invention provides, in one embodiment, a functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0032] This invention provides, in one embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca ⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3. In one embodiment, expression of the isolated nucleic acid is under control of a tissue-specific promoter.

[0033] This invention provides, in one embodiment, a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca ⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3. In one embodiment, the vector comprises a promoter which is regulatable. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0034] This invention provides, in one embodiment, a polypeptide encoded by an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca ⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 8-10. [0035] This invention provides, in one embodiment, an isolated polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said isolated polypeptide has a sequence sharing at least 95% homology with SEQ ID NOs: 8-10.

[0036] In one embodiment, this invention provides an isolated nucleic acid comprising a microRNA. In one embodiment, the microRNA comprises a sequence complementary to a fragment of SEQ ID NOs: 1-3. In one embodiment, the microRNA has a sequence sharing at least 95% homology with SEQ ID NO: 6.

[0037] This invention provides, in one embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13. In one embodiment, expression of the nucleic acid is under control of a tissue-specific promoter.

[0038] This invention provides, in one embodiment, a vector comprising an isolated nucleic acid comprising a nucleic acid sequence encoding a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13. In one embodiment, the vector comprises a promoter which is regulatable. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector. In another embodiment, this invention provides a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13.

[0039] This invention provides, in one embodiment, an isolated polypeptide comprising an amino acid sequence of a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 14.

[0040] In one embodiment, the invention provides an antibody which specifically recognizes a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said isolated polypeptide has a sequence sharing at least 95% homology with SEQ ID NOs: 8-10. In one embodiment, the antibody diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity.

[0041] This invention provides, in one embodiment, a method of treating a disease, which is improved by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby treating said disease. In one embodiment, the disease is a preneoplastic, neoplastic, autoimmune, or reactive cell disorder. In one embodiment, the disease is a cardiovascular disease. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting stimulates or increases expression of said exchanger.

[0042] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0043] This invention provides, in one embodiment, a method of treating a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX \ exchanger expression, activity, function, or a combination thereof, thereby treating said .disease. In one embodiment, the disease is a preneoplastic, neoplastic, autoimmune, or reactive cell disorder. In one embodiment, the disease is a cardiovascular disease. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting stimulates or increases expression of said exchanger.

[0044] This invention encompasses methods of stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. This invention also encompasses methods of diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. In one embodiment, the course of cardiovascular diseases or disorders may be altered by compounds and compositions of this invention. In one embodiment, cardiovascular diseases or disorders may be treated by compounds and compositions of this invention. In one embodiment, diseased cells associated with stimulation or acceleration of calcium flux, or a diminishing or abrogation of calcium flux, may be targeted.

[0045] Diseases which may be treated with compounds and compositions of this invention include, but are not limited to, hypertension, hypotension, metabolic syndrome, autoimmune diseases, retinitis pigmentosa and osteoporosis. In one embodiment, these diseases are associated with stimulation or acceleration of calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. In one embodiment, stimulation or acceleration of calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger of the exchanger will lead to an increase in blood pressure (hypertension). In another embodiment, these diseases are associated with a diminishing or abrogation of calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. In one embodiment, a diminishing or abrogation of calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger will lead to a decrease in blood pressure (hypotension). In one embodiment, these diseases are associated with abnormally high levels of apoptosis. [0046] In one embodiment, the invention provides methods, compounds and compositions for treatment of hypertension (Figures 5 and 6), which, in one embodiment, may be high blood pressure. In one embodiment, hypertension may have no specific cause. In another embodiment, hypertension may be associated with another condition, which, in one embodiment, may include kidney disease or tumors such as pheochromocytoma or paraganglioma. Hypertension may, in one embodiment, be a risk factor for conditions including, but not limited to, stroke, heart attack, heart failure, arterial aneurysm, or chronic renal failure. In another embodiment, hypertension may lead to shortened life expectancy.

[0047] In one embodiment, the invention provides methods of treatment of metabolic syndrome. In one embodiment, metabolic syndrome is a combination of medical factors that increase the risk of developing cardiovascular disease and diabetes, which, in one embodiment, may include abdominal obesity (excessive fat tissue in and around the abdomen), atherogenic dyslipidemia (blood fat disorders - high triglycerides, low HDL cholesterol and high LDL cholesterol - that foster plaque buildups in artery walls), elevated blood pressure, insulin resistance or glucose intolerance, prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), or proinflammatory state (e.g., elevated C-reactive protein in the blood). Other conditions associated with metabolic syndrome include, but are not limited to, physical inactivity, aging, hormonal imbalance and genetic predisposition.

[0048] In one embodiment, the invention provides methods of treatment of hypotension. In one embodiment, symptoms of hypotension comprise, but are not limited to, dizziness, fainting (syncope), lack of concentration, blurred vision, nausea, cold and clammy skin, rapid and shallow breathing, fatigue, depression and thirst. In one embodiment, hypotension is associated with pregnancy. In one embodiment, hypotension is associated with medications, which may include, but are not limited to, diuretics; heart medications such as beta blockers; drugs for Parkinson's disease; tricyclic antidepressants; sildenafil, particularly in combination with nitroglycerine; narcotics and alcohol. In one embodiment, such medications are used to reduce blood pressure in hypertensive subjects. In another embodiment, hypotension is associated with heart problems, which may include, but are not limited to, extremely low heart rate (bradycardia), heart valve problems, heart attack and heart failure. In another embodiment, hypotension is associated with endocrine problems, which may include, but are not limited to, an underactive (hypothyroidism) or overactive (hyperthyroidism) thyroid, adrenal insufficiency (Addison's disease), low blood sugar (hypoglycemia) or diabetes. In other embodiments, hypotension is associated with dehydration, blood loss or reduced blood volume (hypovolemia), septicemia, allergic reaction (anaphylaxis) or nutritional deficiencies. In some embodiments, hypotension may be postural or orthostatic hypotension, or postprandial hypotension. In other embodiments, hypotension may be due to nervous system damage (multiple system atrophy with orthostatic hypotension or Shy-Drager syndrome) or neurally-mediated hypotension.

[0049] This invention encompasses methods of inhibiting cellular apoptosis by diminishing or abrogating mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. In some embodiments, inhibition comprises the step of contacting a cell with an agent.

[0050] In one embodiment, the term "diminishing" refers to reducing mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, as determined by fluorescent measurement of Ca²⁺ signals. In one embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 1-10%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 10-20%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 20-30%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 30-40%. In another embodiment, mitochondrial Na⁺/Ca ⁺ NCLX exchanger activity is reduced from about 40-50%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 50-60%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 60-70%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 70-80%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced from about 80-90%. In another embodiment, mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity is reduced by more than 90%. In one embodiment, the term "abrogation" refers to the elimination of mitochondrial Na⁺/Ca² NCLX exchanger activity.

[0051] In one embodiment, the term "agent" refers to any molecule which satisfies the indicated purpose. In some embodiments, "agent" is a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, a functional fragment thereof, a small molecule, or any chemical moiety suitable for the indicated purpose. In some embodiments, the agent is a vector comprising a nucleic acid as described herein.

[0052] By the term "contacting a cell", as used herein, it is meant to include any exposure of a cell to a peptide, nucleic acid, or composition of this invention. Cells may be in direct contact with compounds and compositions of the invention, or exposed indirectly, through methods well described in the art. For example, cells grown in media in vitro, wherein the media are supplemented with any of the peptides, nucleic acids, compounds or compositions of the invention would be an example of a method of contacting a cell, considered a part of this invention. Another example would be oral or parenteral administration of a peptide, nucleic acid, compound or composition, whose administration results in vivo cellular exposure to these compounds, within specific sites within a body. Such administration is also considered as part of this invention, as part of what is meant by the phrase "contacting a cell". [0053] In some embodiments, the agent is a caspase, which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. The caspases are a family of cysteine proteases that cleave C-terminal to an aspartic acid residue in a peptide and are involved in cell death pathways leading to apoptosis (see Martin and Green, Cell 82:349-352 (1995)). The caspases previously were referred to as the "Ice" proteases, based on their homology to the first identified member of the family, the interleukin-1 beta (IL-1 beta)-converting enzyme (Ice), which converts the inactive 33 kiloDalton (kba) form of IL-1 beta to the active 17.5 kDa form. The Ice protease was found to be homologous to the Caenorhabditis elegans ced-3 gene, which is involved in apoptosis during C. elegans development, and transfection experiments showed that expression of Ice in fibroblasts induced apoptosis in the cells (see Martin and Green, supra, 1995). Additional peptides sharing homology with Ice and ced-3 have been identified and are referred to as caspases, each caspase being distinguished by a number.

[0054] In some embodiments, the agent is a null mutant or siRNA sequence of the nucleic acid sequence encoding the mitochondrial Na⁺/Ca²⁺ NCLX exchanger. The term "siRNA" refers to small inhibitory RNA duplexes derived from processing of linear double-stranded RNA that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally between 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. An siRNA may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that expression of the target gene is inhibited. A delivered siRNA can stay within the cytoplasm or nucleus. The siRNA can be delivered to a cell to inhibit expression of an endogenous or exogenous nucleotide sequence, or to affect a specific physiological characteristic not naturally associated with the cell. A description of the mechanisms for siRNA activity, as well as some of its applications are described in Provost et al. (E.M.B.O. J., 2002 Nov. 1; 21(21): 5864-5874); Tabara et al. (Cell 2002, June 28; 109(7):861- 71); Martinez et al. (Cell 2002, Sep. 6; 110(5):563); Hutvagner & Zamore (Science 2002, 297:2056). It is to be understood that siRNAs comprising the microRNAs of the present invention are considered part of the invention.

[0055] The phrase "gene silencing" refers to a process by which the expression of a specific gene product is lessened or attenuated. Gene silencing can take place by a variety of pathways. Unless specified otherwise, as used herein, gene silencing refers to decreases in gene product expression that results from RNA interference (RNAi), a defined, though partially characterized pathway whereby small inhibitory RNA (siRNA) act in concert with host proteins (e.g. the RNA induced silencing complex, RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion. The level of gene silencing can be measured by a variety of means, including, but not limited to, measurement of transcript levels by northern blot analysis, B-DNA techniques, transcription- sensitive reporter constructs, expression profiling (e.g. DNA chips), and related technologies. Alternatively, the level of silencing can be measured by assessing the level of the protein encoded by a specific gene. This can be accomplished by performing a number of studies including western analysis, measuring the levels of expression of a reporter protein that has e.g. fluorescent properties (e.g. GFP) or enzymatic activity (e.g. alkaline phosphatases), or several other procedures.

[0056] In one embodiment, cellular apoptosis is regulated via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. "Cellular apoptosis" is defined herein as a mechanism of programmed cell death, the most common form of physiological (as opposed to pathological) cell death. Apoptosis is an active process requiring metabolic activity by the dying cell; often characterized by shrinkage of the cell, cleavage of the DNA into fragments that give a so-called "laddering pattern" on gels and by condensation and margination of chromatin. Methodology for measuring apoptosis includes, but is not limited to: measurement of DNA fragmentation by pulsed field gel electrophoresis (Belyaev I.Y. and Harms-Ringdahl M., (2002) Radiats Biol Radioecol 42: 279-83) or by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) (Edston E. et al. (2002) Int J Legal Med 116: 22-6), measurement of membrane dielectric changes (Wang X. et al. (2002) Biochim Biophys Acta 1564: 412-20), microscopic examination and confirmation of the presence of characteristic pyknotic nucleii, and others.

[0057] It is one object of the present invention to provide a method for inhibiting cellular apoptosis, the method comprising the step of contacting a cell with an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby inhibiting apoptosis. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13 or a microRNA having a sequence sharing at least 95% homology with SEQ ID NO: 6, wherein said contacting diminishes or abrogates expression, activity, or a combination thereof, of said exchanger.

[0058] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0059] In one embodiment, the agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0060] This invention provides, in one embodiment, a method of inhibiting cellular apoptosis comprising the step of contacting a cell with an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein, in one embodiment, the cell is a cell of the peripheral nervous system (PNS), central nervous system (CNS), immune cell or cardiac cell.

[0061] It is another object of the present invention to provide a method for stimulating or accelerating cellular apoptosis, the method comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression or activity, function, or a combination thereof, thereby stimulating or accelerating apoptosis. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting up-regulates expression, activity, function, or a combination thereof of said exchanger.

[0062] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

63]This invention provides, in one embodiment, a method of stimulating or accelerating cellular apoptosis comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, wherein, in one embodiment, the cell is a preneoplastic cell, a neoplastic cell, an inflammatory cell or an infected cell, or any combination thereof. In one embodiment, the cell is a T helper cell.

[0064] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NO: 13, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NOs: 1-3, wherein said contacting diminishes or abrogates expression of said exchanger. In one embodiment, the microRNA shares at least 95% homology with SEQ ID NO: 6.

[0065] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0066] In one embodiment, the agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger. [0067] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein, in one embodiment, the pathologic condition is a result of AIDS, or a cardiovascular, neurodegenerative, skeletal, inflammatory or infectious disease or disorder.

[0068] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with calcium flux or, in some embodiments, with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition. In one embodiment, the agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ ID NOs: 1-3, or a microRNA comprising a sequence complementary to a fragment of SEQ ID NO: 13, wherein said contacting stimulates or increases expression of said exchanger.

[0069] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0070] In one embodiment, the agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

[0071] This invention provides, in one embodiment, a method of reducing the severity of a pathologic condition associated with calcium flux or, in some embodiments, with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein, in one embodiment, said pathologic condition is a result of a preneoplastic, neoplastic, autoimmune, or reactive cell disorder.

[0072] In one embodiment, the agent is an isolated nucleic acid, wherein the nucleic acid is a vector. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an integrating vector.

[0073] The nucleic acid utilized in methods and compositions of the present invention has, in one embodiment, the sequence: a tggccggcag aaggctgaat ctgcgctggg cactgagtgt gctttgtgtg ctgctaatgg cggagacagt gtctgggact aggggctcgt ctacaggagc tcacattagc ccccagtttc cagcttcagg tgtgaaccag acccccgtgg tagactgccg caaggtgcgt ggcctgaatg tctctgaccg ctgtgacttc atccggacca accctgactg ccacagtgat ggggggtacc tggactacct ggaaggcatc ttctgccact tccctcccag cctcctccct ctggctgtca ctctctacgt ttcctggctg ctctacctgt ttctgattct gggagtcacc gcagccaagt ttttctgccc caacttgtcg gccattccta ccacactgaa gctctcccac aacgtggcag gcgtcacctt cctggcattt gggaatggtg cacctgacat cttcagtgcc ctggtggcct tctctgaccc gcacacagcc ggcctggccc ttggggcact gtttggcgct ggcgtgctgg ttaccacagt ggtggccgga ggcattacca tcctacaccc cttcatggct gcctccaggc ccttcttcag ggacatcgtt ttctacatgg tggctgtgtt cctgaccttc ctcatgctct tccgtggcag ggtcaccctg gcatgggctc tgggttacct gggcttgtat gtgttctatg tggtcactgt gattctctgc acctggatct accaacggca acggagagga tctctgttct gccccatgcc agttactcca gagatcctct cagactccga ggaggaccgg gtatcttcta gtaccaacag ctatgactac ggtgatgagt accggccgct gttcttctac caggagacca cggctcagat cctggtccgg gccctcaatc ccctggatta catgaagtgg agaaggaaat cagcatactg gaaagccctc aaggtgttca agctgcctgt ggagttcctg ctgctcctca cagtccccgt cgtggacccg gacaaggatg accagaactg gaaacggccc ctcaactgtc tgcatctggt tatcagcccc ctggttgtgg tcctgaccct gcagtcgggg acctatggtg tctatgagat aggcggcctc gttcccgtct gggtcgtggt ggtgatcgca ggcacagcct tggcttcagt gacctttttt gccacatctg acagccagcc ccccaggctt cactggctct ttgctttcct gggctttctg accagcgccc tgtggatcaa cgcggccgcc acagag (SEQ ID NO: 1).

[0074] In another embodiment, a nucleic acid utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 1. In another embodiment, the nucleic acid is an isoform of SEQ ID NO: 1. In another embodiment, the nucleic acid is a variant of SEQ ID NO: 1. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 1. In another embodiment, the nucleic acid is a fragment of an isoform of SEQ ID NO: 1. In another embodiment, the nucleic acid is a fragment of a variant of SEQ ID NO: 1.

[0075] The nucleic acid utilized in methods and compositions of the present invention has, in one embodiment, the sequence: atggctgcctccaggcccttcttcagggacatcgttttctacatggtggctgtgttcctgaccttcctcatgctcttccgtggcagggtcaccct ggcatgggctctgggttacctgggcttgtatgtgttctatgtggtcactgtgattctctgcacctggatctaccaacggcaacggagaggatct ctgttctgccccatgccagttactccagagatcctctcagactccgaggaggaccgggtatcttctagtaccaacagctatgactacggtgat gagtaccggccgctgttcttctaccaggagaccacggctcagatcctggtccgggccctcaatcccctggattacatgaagtggagaagga aatcagcatactggaaagccctcaaggtgttcaagctgcctgtggagttcctgctgctcctcacagtccccgtcgtggacccggacaaggat gaccagaactggaaacggcccctcaactgtctgcatctggttatcagccccctggttgtggtcctgaccctgcagtcggggacctatggtgt ctatgagataggcggcctcgttcccgtctgggtcgtggtggtgatcgcaggcacagccttggcttcagtgaccttttttgccacatctgacag ccagccccccaggcttcactggctctttgctttcctgggctttctgaccagcgccctgtggatcaacgcggccgccacagaggtggtgaaca tcttgcggtccctgggtgtggtcttccggctgagcaacactgtgctggggctcacgctgctggcctgggggaacagcattggagatgccttc tcggatttcacactggctcgccagggctacccacggatggcgttctccgcctgctttggcggcatcatcttcaacatcctcgtgggtgtgggg ctgggctgcctgctccagatctcccgaagccacacagaagtgaagctggagccagacggactgctggtgtgggtcctggcaggcgccct ggggctcagcctcgtcttctccctggtctcagtcccattgcagtgcttccagctcagcagagtctatggcttctgcctgctcctcttctacctga acttccttgtcgtggccctcctcactgaatttggagtgattcacctgaaaagcatg (SEQ ID NO: 2).

[0076] In another embodiment, a nucleic acid utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 2. In another embodiment, the nucleic acid is an isoform of SEQ ID NO: 2. In another embodiment, the nucleic acid is a variant of SEQ ID NO: 2. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 2. In another embodiment, the nucleic acid is a fragment of an isoform of SEQ ID NO: 2. In another embodiment, the nucleic acid is a fragment of a variant of SEQ ID NO: 2.

[0077] The nucleic acid utilized in methods and compositions of the present invention has, in one embodiment, the sequence:

atggccggcagaaggctgaatctgcgctgggcactgagtgtgctttgtgtgctgctaatggcggagacagtgtctgggactaggggctcgt ctacaggagctcacattagcccccagtttccagcttcaggtgtgaaccagacccccgtggtagactgccgcaaggtgcgtggcctgaatgt ctctgaccgctgtgacttcatccggaccaaccctgactgccacagtgatggggggtacctggactacctggaaggcatcttctgccacttcc ctcccagcctcctccctctggctgtcactctctacgtttcctggctgctctacctgtttctgattctgggagtcaccgcagccaagtttttctgccc caacttgtcggccattcctaccacactgaagctctcccacaacgtggcaggcgtcaccttcctggcatttgggaatggtgcacctgacatctt cagtgccctggtggccttctctgacecgcacacagccggcctggcccttggggcactgtttggcgctggcgtgctggttaccacagtggtg gccggaggcattaccatcctacaccccttcatggctgcctccaggcccttcttcagggacatcgttttctacatggtggctgtgttcctgacctt cctcatgctcttccgtggcagggtcaccctggcatgggctctgggttacctgggcttgtatgtgttctatgtggtcactgtgattctctgcacctg gatctaccaacggcaacggagaggatctctgttctgccccatgccagttactccagagatcctctcagactccgaggaggaccgggtatctt ctagtaccaacagctatgactacggccatggcagcgatccgggcccgggcccgcagagcccgggcctgcatgaagtggaaaaagaaat tagcattctggaaagcccgcagggcgtgcaggcggcgtgcggcgtgccggcggcgccgcatagcccgcgccgcggcccgggccagg gc (SEQ ID NO: 3).

[0078] In another embodiment, a nucleic acid utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 3. In another embodiment, the nucleic acid is an isoform of SEQ ID NO: 3. In another embodiment, the nucleic acid is a variant of SEQ ID NO: 3. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 3. In another embodiment, the nucleic acid is a fragment of an isoform of SEQ ID NO: 3. In another embodiment, the nucleic acid is a fragment of a variant of SEQ ID NO: 3.

[0079] This invention provides, in one embodiment, a functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, while in another embodiment, the exchanger is non-functional.

[0080] In one embodiment, a mitochondrial Na⁺/Ca²⁺ NCLX exchanger comprises or consists of the nucleic acid sequence: ccagctgtttggaactgagctactgcagaaagggaagtggagagtaagggccaggccccgtgggggcagatggccggcagaaggctg aatctgcgctgggcactgagtgtgctttgtgtgctgctaatggcggagacagtgtctgggactaggggctcgtctacaggagctcacattag cccccagtttccagcttcaggtgtgaaccagacccccgtggtagactgccgcaaggtgcgtggcctgaatgtctctgaccgctgtgacttca tccggaccaaccctgactgccacagtgatggggggtacctggactacctggaaggcatcttctgccacttccctcccagcctcctccctctg gctgtcactctctacgtttcctggctgctctacctgtttctgattctgggagtcaccgcagccaagtttttctgccccaacttgtcggccattccta ccacactgaagctctcccacaacgtggcaggcgtcaccttcctggcatttgggaatggtgcacctgacatcttcagtgccctggtggccttct ctgacccgcacacagccggcctggcccttggggcactgtttggcgctggcgtgctggttaccacagtggtggccggaggcattaccatcct acaccccttcatggctgcctccaggcccttcttcagggacatcgttttctacatggtggctgtgttcctgaccttcctcatgctcttccgtggcag ggtcaccctggcatgggctctgggttacctgggcttgtatgtgttctatgtggtcactgtgattctctgcacctggatctaccaacggcaacgg agaggatctctgttctgccccatgccagttactccagagatcctctcagactccgaggaggaccgggtatcttctagtaccaacagctatgac tacggtgatgagtaccggccgctgttcttctaccaggagaccacggctcagatcctggtccgggccctcaatcccctggattacatgaagtg gagaaggaaatcagcatactggaaagccctcaaggtgttcaagctgcctgtggagttcctgctgctcctcacagtccccgtcgtggacccg gacaaggatgaccagaactggaaacggcccctcaactgtctgcatctggttatcagccccctggttgtggtcctgaccctgcagtcgggga cctatggtgtctatgagataggcggcctcgttcccgtctgggtcgtggtggtgatcgcaggcacagccttggcttcagtgaccttttttgccac atctgacagccagccccccaggcttcactggctctttgctttcctgggctttctgaccagcgccctgtggatcaacgcggccgccacagagg tggtgaacatcttgcggtccctgggtgtggtcttccggctgagcaacactgtgctggggctcacgctgctggcctgggggaacagcattgg agatgccttctcggatttcacactggctcgccagggctacccacggatggcgttctccgcctgctttggcggcatcatcttcaacatcctcgtg ggtgtggggctgggctgcctgctccagatctcccgaagccacacagaagtgaagctggagccagacggactgctggtgtgggtcctggc aggcgccctggggctcagcctcgtcttctccctggtctcagtcccattgcagtgcttccagctcagcagagtctatggcttctgcctgctcctc ttctacctgaacttccttgtcgtggccctcctcactgaatttggagtgattcacctgaaaagcatgtga (SEQ ID NO: 4), which in one embodiment, is the sequence set forth in NCBI's Genbank Accession NO.: AY601759 and others, as will be appreciated by one skilled in the art. In one embodiment, a nucleic acid of the present invention may comprise a mutation in the wild-type NCLX exchanger, or, in another embodiment, a truncation of the wild-type NCLX exchanger, or a combination thereof. In another embodiment, a nucleic acid of the present invention may comprise a mutation of a truncation of the wild-type NCLX exchanger. In another embodiment, a nucleic acid of the present invention may comprise a mutation or truncation of a homolog of the wild-type NCLX exchanger.

[0081] In another embodiment, a NCLX exchanger is a splice variant of the full length NCLX exchanger, which, in one embodiment, comprises or consists of the nucleic acid sequence: ccagctgtttggaactgagctactgcagaaagggaagtggagagtaagggccaggccccgtgggggcagatggccggcagaaggctg aatctgcgctgggcactgagtgtgctttgtgtgctgctaatggcggagacagtgtctgggactaggggctcgtctacaggagctcacattag cccccagtttccagcttcaggtgtgaaccagacccccgtggtagactgccgcaaggtgtgtggcctgaatgtctctgaccgctgtgacttcat ccggaccaaccctgactgccacagtgatggggggtacctggactacctggaaggcatcttctgccacttccctcccagcctcctccctctgg ctgtcactctctacgtttcctggctgctctacctgtttctgattctgggagtcaccgcagccaagtttttctgccccaacttgtcggccatttctacc acactgaagctctcccacaacgtggcaggcgtcaccttcctggcatttgggaatggtgcacctgacatcttcagtgccctggtggccttctct gacccgcacacagccggcctggcccttggggcactgtttggttacctgggcttgtatgtgttctatgtggtcactgtgattctctgcacctggat ctaccaacggcaacggagaggatctctgttctgccccatgccagttactccagagatcctctcagactccgaggaggaccgggtatcttcta ataccaacagctatgactacggtgatgagtaccggccgctgttcttctaccaggagaccacggctcagatcctggtccgggccctcaatccc ctggattacatgaagtggagaaggaaatcagcatactggaaagccctcaaggtgttcaagctgcctgtggagttcctgctgctcctcacagt ccccgtcgtggacccggacaaggatgaccagaactggaaacggcccctcaactgtctgcatctggttatcagccccctggttgtggtcctg accctgcagtcggggacctatggtgtctatgagataggcggcctcgttcccgtctgggtcgtggtggtgatcgcaggcacagccttggcttc agtgaccttttttgccacatctgacagccagccccccaggcttcactggctctttgctttcctgggctttctgaccagcgccctgtggatcaacg cggccgccacagaggtggtgaacatcttgcggtccctgggtgtggtcttccggctgagcaacactgtgctggggctcacgctgctggcctg ggggaacagcattggagatgccttctcggatttcacactggctcgccagggctacccacggatggcgttctccgcctgctttggcggcatca tcttcaacatcctcgtgggtgtggggctgggctgcctgctccagatctcccgaagccacacagaagtgaagctggagccagacggactgc tggtgtgggtcctggcaggcgccctggggctcagcctcgtcttctccctggtctcagtcccattgcagtgcttccagctcagcagagtctatg gcttctgcctgctcctcttctacctgaacttccttgtcgtggccctcctcactgaatttggagtgattcacctgaaaagcatgtga (SEQ ID NO: 5), which in one embodiment, is the sequence set forth in NCBI's Genbank Accession No.: AY601760 and others, as will be appreciated by one skilled in the art. In one embodiment, a nucleic acid of the present invention may comprise a mutation in an NCLX exchanger splice variant, or, in another embodiment, a truncation of an NCLX exchanger splice variant, or a combination thereof. In another embodiment, a nucleic acid of the present invention may comprise a mutation of a truncation of an NCLX exchanger splice variant. In another embodiment, a nucleic acid of the present invention may comprise a mutation or truncation of a homolog of an NCLX exchanger splice variant.

[0082] In some embodiments, the term "nucleic acid" refers to polynucleotide or to oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA) or mimetic thereof. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally- occurring portions, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0083] As will be appreciated by one skilled in the art, a fragment or derivative of a nucleic acid sequence or gene that encodes for a protein or peptide can still function in the same manner as the entire, wild type gene or sequence. Likewise, forms of nucleic acid sequences can have variations as compared to wild-type sequences, nevertheless encoding the protein or peptide of interest, or fragments thereof, retaining wild-type function exhibiting the same biological effect, despite these variations. Each of these represents a separate embodiment of this present invention.

[0084] In one embodiment, the nucleic acid of the invention comprises an amino acid substitution. In one embodiment, the amino acid substitution is conservative. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0085] In order to avoid severely reducing or eliminating biological activity, amino acid residues that are conserved among various species are not altered (except by conservative substitution). Conserved domains and cysteine residues are less likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved) may not be essential for activity and thus are likely to be amenable to alteration.

[0086] The nucleic acids of this invention can be produced by any synthetic or recombinant process such as is well known in the art. Nucleic acids can further be modified to alter biophysical or biological properties by means of techniques known in the art. For example, the nucleic acid can be modified to increase its stability against nucleases (e.g., "end-capping"), or to modify its lipophilicity, solubility, or binding affinity to complementary sequences.

[0087] DNA according to the invention can also be chemically synthesized by methods known in the art. For example, the DNA can be synthesized chemically from the four nucleotides in whole or in part by methods known in the art. DNA can also be synthesized by preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together, by standard methods known in the art. DNA expressing functional homologues of the protein can be prepared from wild-type DNA by site-directed mutagenesis. The DNA obtained can be amplified by methods known in the art. One suitable method is the polymerase chain reaction (PCR) method. Such methods are well known in the art, see for example, U.S. Pat. No.4,683,195, "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); "Current Protocols in Molecular Biology", Ausubel et al., John Wiley and Sons, Baltimore, Maryland (1989); "A Practical Guide to Molecular Cloning", Perbal, B., John Wiley & Sons, New York (1988); "Recombinant DNA", Watson et al., Scientific American Books, New York; "Genome Analysis: A Laboratory Manual Series", Birren et al. (eds) Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "Transcription and Translation" Hames, B.D., and Higgins S.J., eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are well known in the art and are provided for the convenience of the reader. All the information contained in any reference cited herein is to be understood to be incorporated by reference in its entirety.

[0088] The microRNA (miRNA) used in this invention may be obtained by a variety of methods and from a variety of sources. Such methods are well known in the art, see for example, U.S. Pat. 20080026951. The miRNA may be obtained from a biological sample, such as a cell, tissue, or organ. It may be isolated from a biological sample that contains other RNA molecules as well, such as mRNA, tRNA, and/or rRNA. In certain instances, total RNA is first isolated from the sample and then the miRNA is separated from the other RNA, thereby enriching for miRNA. In some embodiments, the miRNA has been isolated away from other RNA to enrich for the mRNA, such that the miRNA is substantially pure, meaning it is at least about 80%, 85%, 90%, 95% pure or more, but less than 100% pure, with respect to other RNA molecules. Alternatively, enrichment of miRNA may be expressed in terms of fold-enrichment. In certain embodiments, miRNA is enriched with respect to the concentration of miRNA in an RNA isolate or the total RNA in the sample. miRNA can be separated from other RNA molecules using methods known to those of ordinary skill in the art. In some embodiments, miRNA are separated from other RNA molecules using chromatography. Gel chromatography can be implemented to isolate miRNA molecules. In certain embodiments, gel chromatography can be performed using a polyacrylamide gel and tube electrophoresis. In other embodiments, miRNA may be detected by in situ hybridization, in situ PCR or microarrays or by employing microarray technology. Methods for modifying nucleic acids to achieve specific purposes are disclosed in the art, for example, in Sambrook et al. (1989). Moreover, the nucleic acid sequences of the invention can include one or more portions of nucleotide sequence that are non-coding for the protein of interest. Variations in the DNA sequences, which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded thereby, are also encompassed in the invention.

[0089] Protein and/or peptide homology for any peptide sequence listed herein may be determined by immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via methods well known to one skilled in the art. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example.

[0090] In some embodiments, a nucleic acid of the present invention encodes a polypeptide of the present invention. In some embodiments, the nucleic acid may have a sequence corresponding to or homologous to any known sequence encoding the polypeptide, or one inducing the same.

[0091] Homology, as used herein, may refer to sequence identity, or may refer to structural identity, or functional identity. By using the term "homology" and other like forms, it is to be understood that any molecule, whether nucleic acid or peptide, that functions similarly, and/or contains sequence identity, and/or is conserved structurally so that it approximates the reference sequence, is to be considered as part of this invention.

[0092] The term "homology", as used herein, when in reference to Na⁺/Ca²⁺ NCLX exchanger polypeptides, indicates a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art.

[0093] In one embodiment, the terms "homology", "homologue" or "homologous", in any instance, indicate that the sequence referred to, whether an amino acid sequence, or a nucleic acid sequence, exhibits at least 70% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 72% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 75% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 77% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 80% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 82% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 85% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 87% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 92% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 95% - 100% correspondence to the indicated sequence. Similarly, as used herein, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined.

[0094] An additional means of determining homology is via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, (Volumes 1-3) Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). For example, methods of hybridization may be carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42 °C in a solution comprising: 10-20% formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

[0095] In one embodiment, "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them, for example splice variants.

[0096] In one embodiment, "isoform" refers to a version of a molecule, for example, a protein, with only slight differences to another isoform of the same protein. In one embodiment, isoforms may be produced from different but related genes, or in another embodiment, may arise from the same gene by alternative splicing. In another embodiment, isoforms are caused by single nucleotide polymorphisms.

[0097] In one embodiment, "fragment" refers to a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide. In another embodiment, fragment refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment is an intrasequential section of the protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional intrasequential section of the protein, peptide or nucleic acid. In another embodiment, the fragment is a functional intrasequential section within the protein, peptide or nucleic acid. In another embodiment, the fragment is an N-terminal functional fragment. In one embodiment, the fragment is a C-terminal functional fragment. In another embodiment, the fragment is an N-terminal functional fragment. In another embodiment, the fragment is a C- terminal functional fragment. In one embodiment, a fragment has 10-20 nucleic or amino acids, while in another embodiment, a fragment has more than 5 nucleic or amino acids, while in another embodiment, a fragment has 100-200 nucleic or amino acids, while in another embodiment, a fragment has 100-500 nucleic or amino acids, while in another embodiment, a fragment has 50-200 nucleic or amino acids, while in another embodiment, a fragment has 10- 250 nucleic or amino acids.

[0098] In some embodiments, a miRNA comprises a sequence complementary to a fragment of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger. In one embodiment, microRNA (miRNA) refers to 19-25 nt non-coding RNAs derived from endogenous genes that act as post-transcriptional regulators of gene expression. In another embodiment, miRNAs are 21-23 nucleotides in length. In one embodiment, miRNAs are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer. In one embodiment, miRNAs assemble in ribonucleoprotein complexes termed miRNPs and, in one embodiment, recognize their target sites by antisense complementarity thereby mediating down-regulation of their target genes. Near-perfect or perfect complementarity between the miRNA and its target site, in one embodiment, results in target mRNA cleavage, whereas, in another embodiment, limited complementarity between the miRNA and the target site results in translational inhibition of the target gene. In one embodiment, deregulation of miRNA expression may be a cause of human disease, and in one embodiment, detection of expression of miRNAs of the present invention is useful as a diagnostic for diseases or disorders characterized by abnormal levels of apoptosis, which in one embodiment, is decreased levels of apoptosis. In one embodiment, miRNAs of the present invention may be useful for tissue engineering via regulated expression of miRNAs in cells or tissue devoid of particular miRNAs. In another embodiment, miRNAs of the present invention may be useful for therapeutic intervention via delivery or transgenic expression of miRNAs. In one embodiment, miRNAs of the present invention may also represent valuable drug targets themselves.

[0099] The microRNA utilized in methods and compositions of the present invention comprises or consists of, in one embodiment, the sequence: AACGGCCACUCAACUGUCUtt (SEQ ID NO: 6) In another embodiment, a nucleic acid utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 6. In another embodiment, the nucleic acid is an isoform of SEQ ID NO: 6. In another embodiment, the nucleic acid is a variant of SEQ ID NO: 6. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 6. In another embodiment, the nucleic acid is a fragment of an isoform of SEQ ID NO: 6. In another embodiment, the nucleic acid is a fragment of a variant of SEQ ID NO: 6.

[00100] The nucleic acid utilized in methods and compositions of the present invention may, in one embodiment, encode a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger which has, in one embodiment, the sequence: a tggccggcag aaggctgaat ctgcgctggg cactgagtgt gctttgtgtg ctgctaatgg cggagacagt gtctgggact aggggctcgt ctacaggagc tcacattagc ccccagtttc cagcttcagg tgtgaaccag acccccgtgg tagactgccg caaggtgcgt ggcctgaatg tctctgaccg ctgtgacttc atccggacca accctgactg ccacagtgat ggggggtacc tggactacct ggaaggcatc ttctgccact tccctcccag cctcctccct ctggctgtca ctctctacgt ttcctggctg ctctacctgt ttctgattct gggagtcacc gcagccaagt ttttctgccc caacttgtcg gccattccta ccacactgaa gctctcccac aacgtggcag gcgtcacctt cctggcattt gggaatggtg cacctgacat cttcagtgcc ctggtggcct tctctgaccc gcacacagcc ggcctggccc ttggggcact gtttggcgct ggcgtgctgg ttaccacagt ggtggccgga ggcattacca tcctacaccc cttcatggct gcctccaggc ccttcttcag ggacatcgtt ttctacatgg tggctgtgtt cctgaccttc ctcatgctct tccgtggcag ggtcaccctg gcatgggctc tgggttacct gggcttgtat gtgttctatg tggtcactgt gattctctgc acctggatct accaacggca acggagagga tctctgttct gccccatgcc agttactcca gagatcctct cagactccga ggaggaccgg gtatcttcta gtaccaacag ctatgactac ggtgatgagt accggccgct gttcttctac caggagacca cggctcagat cctggtccgg gccctcaatc ccctggatta catgaagtgg agaaggaaat cagcatactg gaaagccctc aaggtgttca agctgcctgt ggagttcctg ctgctcctca cagtccccgt cgtggacccg gacaaggatg accagaactg gaaacggccc ctcaactgtc tgcatctggt tatcagcccc ctggttgtgg tcctgaccct gcagtcgggg acctatggtg tctatgagat aggcggcctc gttcccgtct gggtcgtggt ggtgatcgca ggcacagcct tggcttcagt gacctttttt gccacatctg acagccagcc ccccaggctt cactggctct ttgctttcct gggctttctg accagcgccc tgtggatcaa cgcggccgcc acagaggtgg tgaacatctt gcggtccctg ggtgtggtct tccggctgag caacactgtg ctggggctca cgctgctggc ctgggggaac accattggag atgccttctc ggatttcaca ctggctcgcc agggctaccc acggatggcg ttctccgcct gctttggcgg catcatcttc aacatcctcg tgggtgtggg gctgggctgc ctgctccaga tctcccgaag ccacacagaa gtgaagctgg agccagacgg actgctggtg tgggtcctgg caggcgccct ggggctcagc ctcgtcttct ccctggtctc agtcccattg cagtgcttcc agctcagcag agtctatggc ttctgcctgc tcctcttcta cctgaacttc cttgtcgtgg ccctcctcac tgaatttgga gtgattcacc tgaaaagcat g (SEQ ID NO: 13).

[00101] In another embodiment, a nucleic acid utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 13. In another embodiment, the nucleic acid is an isoform of SEQ ID NO: 13. In another embodiment, the nucleic acid is a variant of SEQ ID NO: 13. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 13. In another embodiment, the nucleic acid is a fragment of an isoform of SEQ ID NO: 13. In another embodiment, the nucleic acid is a fragment of a variant of SEQ ID NO: 13.

[00102] In some embodiments, the isolated nucleic acid is a vector. By "vector" what is meant is a nucleic acid construct containing a sequence of interest that has been subcloned within the vector, in this case, the nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. To generate the nucleic acid constructs in the context of the present invention, the polynucleotide segments encoding sequences of interest can be ligated into commercially available expression vector systems suitable for transducing mammalian cells and for directing the expression of recombinant products within the transduced cells. It will be appreciated that such commercially available vector systems can easily be modified via commonly used recombinant techniques in order to replace, duplicate or mutate existing promoter or enhancer sequences and/or introduce any additional polynucleotide sequences such as for example, sequences encoding additional selection markers or sequences encoding reporter genes.

[00103] A vector according to the present invention may include an appropriate selectable marker. The vector may further include an origin of replication, and may be a shuttle vector, which can propagate both in bacteria, such as, for example, E. coli (wherein the vector comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in vertebrate cells, or integration in the genome of an organism of choice. The vector according to this aspect of the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

[00104] According to another embodiment, nucleic acid vectors comprising the isolated nucleic acid sequences encoding for the protein of interest include a regulatory element, such as a promoter for regulating expression of the isolated nucleic acid. Such promoters are known to be ds-acting sequence elements required for transcription as they serve to bind DNA-dependent RNA polymerase, which transcribes sequences present downstream thereof.

[00105] The nucleic acid vector may be introduced into desired cells by direct DNA uptake techniques, and virus, plasmid, linear DNA or liposome mediated transduction, receptor- mediated uptake and magnetoporation methods employing calcium-phosphate mediated and DEAE-dextran mediated methods of introduction, electroporation, liposome-mediated transfection, direct injection, and receptor-mediated uptake (for further detail see, for example, "Methods in Enzymology" Vol. 1-317, Academic Press, Current Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene Publishing Associates, (1989) and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), or other standard laboratory manuals). [00106] In another embodiment, the vectors may be administered in vitro or ex vivo, for example, for stimulation of calcium flux or cellular apoptosis. In another embodiment, the compounds may be administered in vitro or ex vivo, for example, for stimulation of calcium flux or cellular apoptosis. In another embodiment, the compositions may be administered in vitro or ex vivo, for example, for stimulation of calcium flux or cellular apoptosis. In another embodiment, the vectors/compounds/compositions may be administered in vivo in a number of ways, which are well known in the art. For example, administration may be done topically (including opthalmically, vaginally, rectally, intranasally, in the ear), orally, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intracardiac or intramuscular injection.The vector may, in one embodiment, be an integrating vector. In one embodiment, the term "integrating vector" refers to a vector which integrates or is inserted into a nucleic acid (e.g., a chromosome), which, in one embodiment, is accomplished via an integrase. In one embodiment, integration may be via homologous recombination. In another embodiment, integration is via a transposon. Examples of "integrating vectors" include, but are not limited to, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex viruses, Semliki forest virus, vaccinia viruses, and combinations thereof.

[00107] The vector may, in one embodiment, comprise a regulatable promoter. A regulatable promoter, in one embodiment, is a promoter where the rate of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include compositions, light, heat, stress and the like. In one embodiment, a regulatable promoter may be an inducible promoter, which may, in one embodiment, respond to heavy metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al. Nature (1982), 296, 39-42), to thermal shocks, to hormones (Lee et al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock et al. Nature (1987), 329, 734-736; Israel and Kaufman, Nucleic Acids Res. (1989), 17, 2589-2604) or to chemical agents of the glucose, lactose, galactose or antibiotic type. Further examples may be found in United States Patent Nos. US 6,214, 620 and 6,506, 379. In another embodiment, a regulatable promoter may be a suppressible promoter, which, in one embodiment, may be a tetracycline-responsive element, while in another embodiment, a regulatable promoter may be a repressible promoter. In another embodiment, a regulatable promoter may be one that expresses the sequences of interest constitutively. In one embodiment, a regulatable promoter may be a tissue-specific promoter. In one embodiment, a tissue-specific promoter directs the gene located 3' to it to be expressed predominantly, if not exclusively in the specific cell type where the promoter expressed its endogenous gene (reviewed by Palmiter et al, Ann. Rev. Genet., 20:465-499 (1986)). In one embodiment, a tissue-specific promoter for use in the present invention is the immunoglobulin promoter described by Brinster et al. (Nature, 306:332-336 (1983)) and Storb et al. (Nature, 310:238-231 (1984)); the elastase-I promoter described by Swift et al. (Cell, 38:639-646 (1984)); the globin promoter described by Townes et al. (Mol. Cell. Biol., 5:1977-1983 (1985)), and Magram et al. (Mol. Cell. Biol., 9:4581-4584 (1989)); the insulin promoter described by Bucchini et al. (Proc. Natl. Acad. Sci., USA, 83:2511-2515 (1986)) and Edwards et al. (Cell, 58:161 (1989)); the immunoglobulin promoter described by Ruscon et al. (Nature, 314:330-334 (1985)) and Grosscheld et al. (Cell, 38:647-658 (1984)); the alpha actin promoter described by Shani (Mol. Cell. Biol., 6:2624-2631 (1986)); the alpha crystalline promoter described by Overbeek et al. (Proc. Natl. Acad. Sci. USA, 82:7815-7819 (1985)); the prolactin promoter described by Crenshaw et al. (Genes and Development, 3:959-972 (1989)); the proopiomelanocortin promoter described by Tremblay et al. (Proc. Natl. Acad. Sci., USA, 85:8890-8894 (1988)); the beta thyroid stimulating hormone (BTSH) promoter described by Tatsumi et al. (Nippon Rinsho, 47:2213-2220 (1989)); the mouse mammary tumor virus (MMTV) promoter described by Muller et al. (Cell, 54:105 (1988)); the albumin promoter described by Palmiter et al. (Ann. Rev. Genet., 20:465-499 (1986)); the Keratin promoter described by Vassar et al. (Proc. Natl. Acad. Sci., USA, 86:8565-8569 (1989)); the osteonectin promoter described by McVey et al. (J. Biol. Chem., 263:11,111-11,116 (1988)); the prostate- specific promoter described by Allison et al. (Mol. Cell. Biol., 9:2254-2257 (1989)); the opsin promoter described by Nathans et al. (Proc. Natl. Acad. Sci., USA, 81:4851-4855 (1984)); the olfactory marker protein promoter described by Danciger et al. (Proc. Natl. Acad. Sci., USA, 86:8565-8569 (1989)); the neuron-specific enolase (NSE) promoter described by Forss-Pelter et al. (J. Neurosci. Res., 16:141-151 (1986)); the L-7 promoter described by Sutcliffe (Trends in Genetics, 3:73-76 (1987)), the protamine 1 promoter described Peschon et al. (Ann. New York Acad. Sci., 564:186-197 (1989)) and Braun et al. (Genes and Development, 3:793-802 (1989)), or a combination thereof.

[00108] Nucleotide sequences which regulate expression of a gene product, which are, in one embodiment, promoter and enhancer sequences, are selected, in another embodiment, based upon the type of cell in which the gene product is to be expressed (tissue-specific promoter), or, in another embodiment, upon the desired level of expression of the gene product, in cells infected with the vectors of the invention. According to this aspect of the invention, the gene product corresponds to the heterologous protein, as described herein. Regulated expression of such a heterologous protein may thus be accomplished, in one embodiment.

[00109] For example, a promoter known to confer cell type-specific expression of a gene linked to the promoter can be used. In another embodiment, a regulatory element, which can direct constitutive expression of a gene in a variety of different cell types, such as a viral regulatory element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40, and retroviral LTRs.

[00110] In another embodiment, a regulatory element, which provides inducible expression of a gene linked thereto can be used. The use of an inducible regulatory element (e.g., an inducible promoter) allows for modulation of the production of the gene product in the cell. In another embodiment, the inducible regulatory systems for use in eukaryotic cells include hormone- regulated elements (e.g., see Mader, S. and White, J.H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see, e.g., Spencer, D.M. et al. 1993) Science 262:1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Additional tissue-specific or inducible regulatory systems may be developed for use in accordance with the invention.

[00111] A vector according to the present invention, may, in another embodiment further include an appropriate selectable marker. The vector may further include an origin of replication, and may be a shuttle vector, which can propagate both in prokaryotic, and in eukaryotic cells, or the vector may be constructed to facilitate its integration within the genome of an organism of choice. The vector, in other embodiments may be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome. In another embodiment, the vector is a viral particle comprising the nucleic acids of the present invention. In one embodiment, the vector of the present invention is a viral vector. In one embodiment, some or all of the viral genes of the viral vector have been replaced by a gene that is to be expressed in the eukaryotic target cell. The essential viral genes that have been removed from the vector are, in general, inserted into the genome of the cell line that is used to produce stocks of the viral particles. The producer cells lines thus complement the defects that are present in the viral vector. In some embodiments, the only viral gene contained in the genome of the vector is a gene that is needed for the packaging of the vector genome into the viral particles. Examples of viral vectors include, but are not limited to, viruses such as herpes virus, retrovirus, vaccinia virus and adenovirus.

[00112] In another embodiment, this invention provides liposomes comprising the nucleic acids and vectors of this invention. Methods for preparing such liposomes are well known in the art, and may be as described in, for example WO 96/18372; WO 93/24640; Mannino and Gould- Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7414).

[00113] The term "peptide", when in reference to any peptide of this invention, is meant to include native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[00114] It is to be understood that any amino acid sequence, whether obtained naturally or synthetically, by any means, exhibiting sequence, structural, or functional homology to the peptides described herein, are considered as part of this invention.

[00115] In some embodiments, the polypeptide utilized in methods and compositions of the present invention is encoded by the nucleic acid sequence encoding the mitochondrial Na⁺/Ca²⁺ NCLX exchanger itself or a functional fragment or derivative thereof. In some embodiments, the polypeptide may have a sequence corresponding to or homologous to any known sequence for the protein, or one inducing the same. In some embodiments, the present invention describes an antibody which recognizes a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity.

[00116] The polypeptide utilized in methods and compositions of the present invention has, in one embodiment, the sequence:

MAGRRLNLRWALSVLCVLLMAETVSGTRGSSTGAHISPQFPASGVNQTPVVDCR KVRGLNVSDRCDFIRTNPDCHSDGGYLDYLEGIFCHFPPSLLPLAVTLYVSWLLYLFLIL GVTAAKFFCPNLSAIPTTLKLSHNVAGVTFLAFGNGAPDIFSALVAFSDPHTAGLALGAL FGAGVLVTTVVAGGITILHPFMAASRPFFRDIVFYMVAVFLTFLMLFRGRVTLAWALGY LGLYVFYVVTVILCTWIYQRQRRGSLFCPMPVTPEILSDSEEDRVSSSTNSYDYGDEYRP LFFYQETTAQILVRALNPLDYMKWRRKSAYWKALKVFKLPVEFLLLLTVPVVDPDKDD QNWKRPLNCLHLVISPLVVVLTLQSGTYGVYEIGGLVPVWVVVVIAGT ALAS VTFF ATS DSQPPRLHWLFAFLGFLTSALWINAAATE (SEQ ID NO: 8), which in one embodiment, represents amino acids 1-439 of the native NCLX exchanger. In another embodiment, a polypeptide utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 8. In another embodiment, the polypeptide is an isoform of SEQ ID NO: 8. In another embodiment, the protein is a variant of SEQ ID NO: 8. In another embodiment, the protein is a fragment of SEQ ID NO: 8. In another embodiment, the protein is a fragment of an isoform of SEQ ID NO: 8. In another embodiment, the protein is a fragment of a variant of SEQ ID NO: 8.

[00117] The polypeptide utilized in methods and compositions of the present invention has, in one embodiment, the sequence: MAASRPFFRDIVFYMVAVFLTFLMLFRGRVTLAWALGYLGLYVFYVVTVILCTWIYQR QRRGSLFCPMPVTPEILSDSEEDRVSSSTNSYDYGDEYRPLFFYQETTAQILVRALNPLDY MKWRRKSAYWKALKVFKLPVEFLLLLTVPVVDPDKDDONWKRPLNCLHLVISPLVVV LTLOSGTYGVYEIGGLVPVWVVVVIAGTALASVTFFATSDSQPPRLHWLFAFLGFLTSA LWINAAATCVVNILRSLGVVFR115NTVLGLTLIAWGNSIGDAPSDFTI_ARQGYPRMAFS ACFGGIIFNILVGVGLGCLLQISRSHTEVKLEPDGLLVWVLAGALGLSLVFSLVSVPLQCF QLS R V YGFCLLLFYLNFLV V ALLTEFG VIHLKS M (SEQ ID NO: 9), which in one embodiment, represents amino acids 196-584 of the native NCLX exchanger. In another embodiment, a polypeptide utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 9. In another embodiment, the polypeptide is an isoform of SEQ ID NO: 9. In another embodiment, the protein is a variant of SEQ ID NO: 9. In another embodiment, the protein is a fragment of SEQ ID NO: 9. In another embodiment, the protein is a fragment of an isoform of SEQ ID NO: 9. In another embodiment, the protein is a fragment of a variant of SEQ ID NO: 9

[00118] The polypeptide utilized in methods and compositions of the present invention has, in one embodiment, the sequence:

MAGRRLNLRWALSVLCVLLMAETVSGTRGSSTGAHISPQFPASGVNQTPVVDCR KVRGLNVSDRCDFIRTNPDCHSDGGYLDYLEGIFCHFPPSLLPLAVTLYVSWLLYLFLIL GVTAAKFFCPNI_^AIPTTLKI_SHNVAGVTFIAFGNGAPDIFSALVAFSDPHTAGI-ALGAL FGAGVLVTTVVAGGITILHPFMAASRPFFRDIVFYMVAVFLTFLMLFRGRVTLAWALGY LGLYVFYVVTVILCTWIYQRQRRGSLFCPMPVTPEILSDSEEDRVSSSTNSYDYGHGSDP GPGPQSPGLHEVEKEISILESPQGVQAACGVPAAPHSPRRGPGQG (SEQ ID NO: 10), which in one embodiment, is referred to as NCLX_1.2, which in one embodiment, comprises amino acids 1-287 of native NCLX isoform and a 45 amino acid extension (SEQ ID NO: 10).

[00119] In another embodiment, a polypeptide utilized in methods and compositions of the present invention is a homologue of SEQ ID NO: 10. In another embodiment, the polypeptide is an isoform of SEQ ID NO: 10. In another embodiment, the protein is a variant of SEQ ID NO: 10. In another embodiment, the protein is a fragment of SEQ ID NO: 10. In another embodiment, the protein is a fragment of an isoform of SEQ ID NO: 10. In another embodiment, the protein is a fragment of a variant of SEQ ID NO: 10. [00120] In one embodiment, a polypeptide utilized in methods and compositions of the present invention is a native isoform with deletion of 35 bp, resulting in frameshift and a deletion of the entire a2 domain.

[00121] In one embodiment, a mitochondrial Na⁺/Ca²⁺ NCLX exchanger comprises or consists of the amino acid sequence:

MAGRRLNLRWALSVLCVLLMAETVSGTRGSSTGAHISPQFPASGVNQTPVVDCRKVRG LNVSDRCDFIRTNPDCHSDGGYLDYLEGIFCHFPPSLLPLAVTLYVSWLLYLFLILGVTA AKFFCPNI^AIPT LKLSHNVAGVTFLAFGNGAPDIFSALVAFSDPHTAGLALGALFGAG VLVTTVVAGGITILHPF AASRPFFRDIVFYMVAVFLTFLMLFRGRVTLAWALGYLGLY VFYVVTVILCTWIYQRQRRGSLFCP PVTPEILSDSEEDRVSSSTNSYDYGDEYRPLFFY QETTAQILVRALNPLDYMKWRRKSAYWKALKVFKLPVEFLLLLTVPVVDPDKDDQNW KRPLNCLHLVISPLVVVLTLQSGTYGVYEIGGLVPVWVVVVIAGTALASVTFFATSDSQP PRLHWLFAFLGFLTSALWINAAATEVVNILRSLGVVFRLSNTVLGLTLLAWGNSIGDAFS DFTLARQGYPRMAFSACFGGIIFNILVGVGLGCLLQISRSHTEVKLEPDGLLVWVLAGAL GLSLVFSLVSVPLQCFOLSRVYGFCLLLFYLNFLWALLTEFGVIHLKSM (SEQ ID NO: 11), which in one embodiment, is the sequence set forth in NCBI's Genbank Accession No.: AAT35807 and others, as will be appreciated by one skilled in the art. In one embodiment, SEQ ID NO: 11 is the sequence of the wild-type NCLX exchanger, and in another embodiment, the full length NCLX exchanger. In one embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation in the wild-type NCLX exchanger, or, in another embodiment, a truncation of the wild-type NCLX exchanger, or a combination thereof. In one embodiment, a mutation comprises an insertion, deletion, truncation, or substitution. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation of a truncation of the wild-type NCLX exchanger. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation or truncation of a homolog of the wild-type NCLX exchanger.

[00122] In another embodiment, a NCLX exchanger is a splice variant of the full length NCLX exchanger, which, in one embodiment, comprises or consists of the amino acid sequence:

MAGRRLNLRWALSVLCVLLMAETVSGTRGSSTGAHISPQFPASGVNQTPVVDCR KVCGLNVSDRCDFIRTNPDCHSDGGYLDYLEGIFCHFPPSLLPLAVTLYVSWLLYLFLIL GVTAAKPFCPNL^AISTTLKl^HNVAGVTFLAFGNGAPDIFSALVAFSDPHTAGLALGAL FGYLGLYVFYVVTVILCTWIYQRQRRGSLFCPMPVTPEILSDSEEDRVSSNTNSYDYGDE YRPIJFYQETTAQILVRALNPLDY KWRRKSAYWKALKVFKLPVEFLLLLTVPVVDPD KDDQNWKRPLNCLHLVISPLVVVLTLQSGTYGVYEIGGLVPVWVVVVIAGTALASVTF FATSDSQPPRUTWLFAFLGFLTSALWINAAATEVVNILRSLGVVFRLSNTVLGLTLLAW GNSIGDAFSDFTLAROGYPRMAFSACFGGIIFNILVGVGLGCLLQISRSHTEVKLEPDGLL VWVLAGALGLSLVFSLVSVPLQCFQLSRVYGFCLLLFYLNFLVVALLTEFGVIHLKSM

(SEQ ID NO: 12), which in one embodiment, is a short native isoform of NCLX, which in one embodiment, is referred to as s-NCLX, which in one embodiment, is the sequence set forth in Genbank Accession Number AAT35808. In one embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation in SEQ ID NO: 12, or, in another embodiment, a truncation of SEQ ID NO: 12, or a combination thereof. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation of a truncation of SEQ ID NO: 12. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise a mutation or truncation of a homolog of SEQ NO: 12. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may be a substitution or insertion mutant. In another embodiment, a polypeptide utilized in methods and compositions of the present invention may comprise one or more mutations, substitutions, insertions, deletions, or truncations of other splice variants of the NCLX exchanger known in the art.

[00123] The polypeptide utilized in methods and compositions of the present invention may, in one embodiment, be a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger which has, in one embodiment, the sequence:

MAGRRLNLRWALSVLCVLLMAETVSGTRGSSTGAHISPQFPASGVNQTPVVDCR KVRGLNVSDRCDFIRTNPDCHSDGGYLDYLEGIFCHFPPSLLPLAVTLYVSWLLYLFLIL GVTAAKFFCPNLSAIPTTLKLSHNVAGVTFLAFGNGAPDIFSALVAFSDPHTAGLALGAL FGAGVLVTTVVAGGITILHPFMAASRPFFRDIVFYMVAVFLTFLMLFRGRVTLAWALGY LGLYVFYVVTVILCTWIYQRQRRGSLFCPMPVTPEILSDSEEDRVSSSTNSYDYGDEYRP LFFYQETTAQILVRALNPLDYMKWRRKSAYWKALKVFKLPVEFLLLLTVPVVDPDKDD QNWKRPLNCLHLVISPLVVVLTLQSGTYGVYEIGGLVPVWVVVVIAGTALASVTFFATS DSQPPRLHWLFAFLGFLTSALWINAAATEVVNILRSLGVVFRLSNTVLGLTLLAWGNTI GDAFSDFTLARQGYPRMAFSACFGGIIFNILVGVGLGCLLQISRSHTEVKLEPDGLLVWV LAGALGLSLVFSLVSVPLQCFQLSRVYGFCLLLFYLNFLVVALLTEFGVIHLKSM (SEQ NO: 14).

[00124] In another embodiment, a polypeptide utilized in methods and compositions of the present invention is a homologue of SEQ NO: 14. In another embodiment, the polypeptide is an isoform of SEQ NO: 14. In another embodiment, the protein is a variant of SEQ NO: 14. In another embodiment, the protein is a fragment of SEQ NO: 14. In another embodiment, the protein is a fragment of an isoform of SEQ NO: 14. In another embodiment, the protein is a fragment of a variant of SEQ NO: 14.

[00125] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site, which specifically binds an antigen. A molecule which specifically binds a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity does not substantially bind other molecules in a sample. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.

[00126] It is to be understood that this invention provides compositions, kits and uses of any combination of any agents as described herein, and such combinations represent embodiments of this invention.

[00127] In some embodiments, inhibition of cellular apoptosis comprises the step of contacting a cell, which, in some embodiments, is a cell of the peripheral nervous system (PNS), central nervous system (CNS), immune cell or cardiac cell, or any combination thereof.

[00128] This invention encompasses administration of compounds as described herein or compositions comprising the same, for treating diseases and disorders related to degenerative or atrophic conditions of the CNS, diseases and disorders of the immune system or diseases and disorders of the cardiovascular system, or any combination thereof.

[00129] Drug delivery to the CNS may, in some embodiments of this invention, be by systemic administration, injection into CSF pathways, or direct injection into the brain, and in some embodiments, the compositions of this invention are formulated for any of these routes. In one embodiment, the compositions of the present invention are administered by systemic or direct administration into the CNS for targeted action in the CNS, and in some embodiments, the compositions of this invention are formulated for any of these routes. In one embodiment, the composition as set forth herein is formulated for brain-specific delivery, and in some embodiments, the compositions of this invention are formulated for any of these routes. In one embodiment, strategies for drug delivery to the brain include osmotic and chemical opening of the blood-brain barrier (BBB), as well as the use of transport or carrier systems, enzymes, and receptors that control the penetration of molecules in the blood-brain barrier endothelium, and in some embodiments, the compositions of this invention are formulated for any of these routes. In another embodiment, receptor-mediated transcytosis can transport peptides and proteins across the BBB, and in some embodiments, the compositions of this invention are formulated for any of these routes. In other embodiments, strategies for drug delivery to the brain involve bypassing the BBB, and in some embodiments, the compositions of this invention are formulated for any of these routes. In some embodiments, various pharmacological agents are used to open the BBB, and in some embodiments, the compositions of this invention are formulated for any of these routes.

[00130] In one embodiment, the route of administration may be directed to an organ or system that is affected by neurodegenerative conditions. For example, compounds may be administered topically. In another embodiment, the route of administration may be directed to a different organ or system than the one that is affected by neurodegenerative conditions. For example, compounds may be administered parenterally to treat neurodegenerative conditions. Thus, the present invention provides for the use of various dosage forms suitable for administration using any of the routes listed herein, and any routes which avail the CNS of such materials, as will be appreciated by one skilled in the art.

[00131] In some embodiments, the compositions/agents of the invention are specifically formulated such that they cross the blood-brain barrier (BBB). One example of such formulation comprises the use of specialized liposomes, which may be manufactured, for example as described U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. In some embodiments, the liposomes comprise one or more moieties which are selectively transported into specific cells or organs ("targeting moieties" or "targeting groups" or "transporting vectors"), thus providing targeted drug delivery (see, e.g., V. V. Ranade, J. Clin. Phamacol. 29, 685 (1989) fully incorporated by reference herein). In some embodiments the agents are linked to targeting groups that facilitate penetration of the blood brain barrier. In some embodiments, to facilitate transport of agents of the invention across the BBB, they may be coupled to a BBB transport vector (see, for example, Bickel et al, Adv. Drug Delivery Reviews 46:247-79 (2001) fully incorporated by reference herein). In some embodiments, transport vectors include cationized albumin or the 0X26 monoclonal antibody to the transferrin receptor; which undergo absorptive.- mediated and receptor-mediated transcytosis through the BBB, respectively. Natural cell metabolites that may be used as targeting groups include, inter alia, putrescine, spermidine, spermine, or DHA. Other exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 fully incorporated by reference herein); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153:1038 (1988) fully incorporated by reference herein); antibodies (P.G. Bloeman et al, FEBS Lett. 357:140 (1995); M. Owais et al, Antimicrob. Agents Chemother. 39:180 (1995)); surfactant protein A receptor (Briscoe et al, Am. J. Physiol. 1233:134 (1995 fully incorporated by reference herein)); gpl20 (Schreier et al, J. Biol. Chem. 269:9090 (1994)); see also, K. Keinanen and M. L. Laukkanen, FEBS Lett. 346:123 (1994); J.J. Killion and I.J. Fidler, Immunomethods 4:273 (1994) all of which are fully incorporated by reference herein). [00132] In some embodiments, BBB transport vectors that target receptor-mediated transport systems into the brain comprise factors such as insulin, insulin-like growth factors ("IGF-I," and "IGF-II"), angiotensin II, atrial and brain natriuretic peptide ("ANP," and "BNP"), interleukin I ("IL-1") and transferrin. Monoclonal antibodies to the receptors that bind these factors may also be used as BBB transport vectors. BBB transport vectors targeting mechanisms for absorptive- mediated transcytosis include cationic moieties such as cationized LDL, albumin or horseradish peroxidase coupled with polylysine, cationized albumin or cationized immunoglobulins. Small basic oligopeptides such as the dynorphin analogue E-2078 and the ACTH analogue ebiratide may also cross the brain via absorptive -mediated transcytosis and are potential transport vectors. Other BBB transport vectors target systems for transporting nutrients into the brain. Examples of such BBB transport vectors include hexose moieties, e.g., glucose and monocarboxylic acids, e.g., lactic acid and neutral amino acids, e.g., phenylalanine and amines, e.g., choline and basic amino acids, e.g., arginine, nucleosides, e.g., adenosine and purine bases, e.g., adenine, and thyroid hormone, e.g., triiodothyridine. Antibodies to the extracellular domain of nutrient transporters may also be used as transport vectors. Other possible vectors include angiotensin II and ANP, which may be involved in regulating BBB permeability.

[00133] In some cases, the bond linking the therapeutic agent to the transport vector may be cleaved following transport into the brain in order to liberate the biologically active agent. Exemplary linkers include disulfide bonds, ester-based linkages, thioether linkages, amide bonds, acid-labile linkages, and Schiff base linkages. Avidin/biotin linkers, in which avidin is covalently coupled to the BBB drug transport vector, may also be used. Avidin itself may be a drug transport vector. Transcytosis, including receptor-mediated transport of compositions across the blood brain barrier, may also be suitable for the agents of the invention. Transferrin receptor- mediated delivery is disclosed in U.S. Pat. Nos. 5,672,683; 5,383,988; 5,527,527; 5,977,307; and 6,015,555, all of which are fully incorporated herein by reference. Transferrin-mediated transport is also known. P.M. Friden et al, Pharmacol. Exp. Ther. 278:1491-98 (1996); H.J. Lee, J. Pharmacol. Exp. Ther. 292:1048-52 (2000) all of which are fully incorporated herein by reference. EGF receptor-mediated delivery is disclosed in Y. Deguchi et al., Bioconjug. Chem. 10:32-37 (1999), and transcytosis is described in A. Cerletti et al., J. Drug Target 8:435-46 (2000) all of which are fully incorporated herein by reference. Insulin fragments have also been used as carriers for delivery across the blood brain barrier. M. Fukuta et al., Pharm. Res. 11:1681-88 (1994). Delivery of agents via a conjugate of neutral avidin and cationized human albumin has also been described. Y.S. Kang et al., Pharm. Res. 1:1257-64 (1994) all of which are fully incorporated herein by reference. Other modifications in order to enhance penetration of the agents of the invention across the blood brain barrier may be accomplished using methods and derivatives known in the art. For example, U.S. Pat. No. 6,024,977 discloses covalent polar lipid conjugates for targeting to brain and central nervous system. U.S. Pat. No. 5,017,566 discloses cyclodextrin derivatives comprising inclusion complexes of lipoidal forms of dihydropyridine redox targeting moieties. U.S. Pat. No. 5,023,252 discloses the use of pharmaceutical compositions comprising a neurologically active drug and a compound for facilitating transport of the drug across the blood-brain barrier including a macrocyclic ester, diester, amide, diamide, amidine, diamidine, thioester, dithioester, thioamide, ketone or lactone. U.S. Pat. No. 5,024,998 discloses parenteral solutions of aqueous-insoluble drugs with cyclodextrin derivatives. U.S. Pat. No. 5,039,794 discloses the use of a metastatic tumor-derived egress factor for facilitating the transport of compounds across the blood-brain barrier. U.S. Pat. No. 5,112,863 discloses the use of N-acyl amino acid derivatives as antipsychotic drugs for delivery across the blood-brain barrier. U.S. Pat. No. 5,124,146 discloses a method for delivery of therapeutic agents across the blood-brain barrier at sites of increase permeability associated with brain lesions. U.S. Pat. No. 5,153,179 discloses acylated glycerol and derivatives for use in a medicament for improved penetration of cell membranes. U.S. Pat. No. 5,177,064 discloses the use of lipoidal phosphonate derivatives of nucleoside antiviral agents for delivery across the blood-brain barrier. U.S. Pat. No. 5,254,342 discloses receptor-mediated transcytosis of the blood-brain barrier using the transferrin receptor in combination with pharmaceutical compounds that enhance or accelerate this process. U.S. Pat. No. 5,258,402 discloses treatment of epilepsy with imidate derivatives of anticonvulsive sulfamate. U.S. Pat. No. 5,270,312 discloses substituted piperazines as central nervous system agents. U.S. Pat. No. 5,284,876 discloses fatty acid conjugates of dopamine drugs. U.S. Pat. No. 5,389,623 discloses the use of lipid dihydropyridine derivatives of anti-inflammatory steroids or steroid sex hormones for delivery across the blood-brain barrier. U.S. Pat. No. 5,405,834 discloses prodrug derivatives of thyrotropin releasing hormone. U.S. Pat. No. 5,413,996 discloses acyloxyalkyl phosphonate conjugates of neurologically-active drugs for anionic sequestration of such drugs in brain tissue. U.S. Pat. No. 5,434,137 discloses methods for the selective opening of abnormal brain tissue capillaries using bradykinin infused into the carotid artery. U.S. Pat. No. 5,442,043 discloses a peptide conjugate between a peptide having a biological activity and incapable of crossing the blood-brain barrier and a peptide which exhibits no biological activity and is capable of passing the blood-brain barrier by receptor-mediated endocytosis. U.S. Pat. No. 5,466,683 discloses water soluble analogues of an anticonvulsant for the treatment of epilepsy. U.S. Pat. No. 5,525,727 discloses compositions for differential uptake and retention in brain tissue comprising a conjugate of a narcotic analgesic and agonists and antagonists thereof with a lipid form of dihydropyridine that forms a redox salt upon uptake across the blood-brain barrier that prevents partitioning back to the systemic circulation all of which are fully incorporated herein by reference.

[00134] It is to be understood that reference to any publication, patent application or issued patent is to be considered as fully incorporated herein by reference in its entirety.

[00135] Nitric oxide is a vasodilator of the peripheral vasculature in normal tissue of the body. Increasing generation of nitric oxide by nitric oxide synthase causes vasodilation without loss of blood pressure. The blood-pressure-independent increase in blood flow through brain tissue increases cerebral bioavailability of blood-born compositions. This increase in nitric oxide may be stimulated by administering L-arginine. As nitric oxide is increased, cerebral blood flow is consequently increased, and drugs in the blood stream are carried along with the increased flow into brain tissue. Therefore, L-arginine may be used in the pharmaceutical compositions of the invention to enhance delivery of agents to brain tissue after introducing a pharmaceutical composition into the blood stream of the subject substantially contemporaneously with a blood flow enhancing amount of L-arginine, as described in WO 00/56328.

[00136] Still further examples of modifications that enhance penetration of the blood-brain barrier are described in International (PCT) Application Publication Number WO 85/02342, which discloses a drug composition comprising a glycerolipid or derivative thereof. PCT Publication Number WO 089/11299 discloses a chemical conjugate of an antibody with an enzyme which is delivered specifically to a brain lesion site for activating a separately-administered neurologically-active prodrug. PCT Publication Number WO 91/04014 discloses methods for delivering therapeutic and diagnostic agents across the blood-brain barrier by encapsulating the drugs in liposomes targeted to brain tissue using transport-specific receptor ligands or antibodies. PCT Publication Number WO 91/04745 discloses transport across the blood-brain barrier using cell adhesion molecules and fragments thereof to increase the permeability of tight junctions in vascular endothelium. PCT Publication Number WO 91/14438 discloses the use of a modified, chimeric monoclonal antibody for facilitating transport of substances across the blood-brain barrier. PCT Publication Number WO 94/01131 discloses lipidized proteins, including antibodies. PCT Publication Number WO 94/03424 discloses the use of amino acid derivatives as drug conjugates for facilitating transport across the blood-brain barrier. PCT Publication Number WO 94/06450 discloses conjugates of neurologically-active drugs with a dihydropyridine-type redox targeting moiety and comprising an amino acid linkage and an aliphatic residue. PCT Publication Number WO 94/02178 discloses antibody-targeted liposomes for delivery across the blood-brain barrier. PCT Publication Number WO 95/07092 discloses the use of drug-growth factor conjugates for delivering drugs across the blood-brain barrier. PCT Publication Number WO 96/00537 discloses polymeric microspheres as injectable drug-delivery vehicles for delivering bioactive agents to sites within the central nervous system. PCT Publication Number WO 96/04001 discloses omega-3-fatty acid conjugates of neurologically- active drugs for brain tissue delivery. PCT WO 96/22303 discloses fatty acid and glycerolipid conjugates of neurologically-active drugs for brain tissue delivery. In one embodiment, the active compound can be delivered in a vesicle, for example, a liposome. In another embodiment, the active compound can be delivered as a nanoparticle. In one embodiment, delivery may be specifically targeted to the CNS. In another embodiment, the active compounds may be delivered by any method described herein. The compositions of this invention may comprise ingredients known to the skilled artisan to be useful in formulating compositions for administration to a subject. In some embodiments, the compositions will comprise pharmaceutically acceptable carriers or diluents. In some embodiments, the phrase "pharmaceutically acceptable carriers or diluents" may comprise a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof.

[00137] In some embodiments, the compositions/agents of the invention comprise a "piggyback mechanism" to deliver specific desirable agents, or combinations thereof to the CNS, i.e. to ensure that they cross the blood-brain barrier (BBB).

[00138] This invention encompasses methods of stimulating or accelerating cellular apoptosis by stimulating or increasing mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. In some embodiments, stimulation or acceleration comprises the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. In some embodiments, stimulation or acceleration of cellular apoptosis comprises the step of contacting a cell, which, in some embodiments, is a preneoplastic cell, a neoplastic cell, an inflammatory cell, an autoimmune reactive cell or an infected cell, or any combination thereof. Inflammatory cells may include, but are not limited to, lymphocytes and monocyte/macrophages. A reactive cell may include antigen-reactive T cells, which may, in some embodiments, be generated by stimulation and proliferation of a subset of T cells. In another embodiment, the reactive cell may be a dendritic cell. In another embodiment, cells may be infected by a virus particle, for example adenovirus or HIV, a parasite, for example malaria, or a bacterium.

[00139] This invention encompasses methods of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject which, in some embodiments, comprises the step of administering an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity.

[00140] Stimulation of apoptosis may be conducted in healthy cells, by the methods disclosed herein, utilizing the aforementioned vectors/compounds/compositions. In another embodiment, the invention provides a method of stimulating apoptosis in a diseased cell. Such stimulation may function as a means of treatment for a given disease, wherein the disease is associated with an abnormally low level of apoptotic cell death. By administering to diseased cells an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, or a pharmaceutical composition comprising an a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger molecule, apoptosis is stimulated, thereby accelerating apoptosis.

[00141] The cell chosen may be any in which stimulation of apoptosis is desirable. In one embodiment, the diseased cell is a preneoplastic cell, a neoplastic cell, an inflammatory cell, an infected cell, or a fat cell. For example, preneoplastic cells in the central nervous system that fail to undergo apoptosis, yet themselves are not neoplastic, still provide a significant threat to an afflicted host, and selected cellular apoptosis is a therapeutic means of reducing an expanding cell mass in a sensitive anatomic region. Similarly, superantigen engagement of T cell receptors is a mechanism whereby T cell proliferation is enhanced, resulting in autoimmune disease and its associated phenomenon. Selective apoptosis of expanded T cells is a means of controlling T cell proliferation, and downstream effects of such cellular expansion. Selective apoptosis of neoplastic cells causing any of many types of cancer is a well-documented therapeutic approach, with numerous applications well known and investigated in the art [see, for example, Tong Y. et al. Mol Cancer Ther. (2001) 1:95-102; Mora L.B. et al. Cancer Res. (2002) 62:6659-66; Opalka B. et al. Cells Tissues Organs. (2002) 172:126-32]. In addition, undesirable expansion of cell populations occurs in conditions such as psoriasis [Laporte M. et al. Dermatology. (2000) 200; 314-6] and in restenosis [Kolesnick R., J Clin Invest. (2002) 110: 3-8], where such therapeutic intervention would be expected to be highly beneficial. It may also be desirable to target infected cells for apoptosis, prior to cell-to-cell spread of a given pathogen. For example, infection with Mycobacterium tuberculosis is known to form a single primary focus of infection, where stimulation of apoptosis within the focus may prevent further dissemination and disease. It may also be desirable to target fat cells, where stimulation of apoptosis may be used to treat obesity. See, for example, WO/2005/082349 and United States Patent 20080015257.

[00142] In one embodiment, the compounds of the present invention are administered to any cell in which stimulation of apoptosis is desirable, such as neoplastic (cancer) cells. In one embodiment, the compound of the present invention is administered in combination with a cancer vaccine. In one embodiment, the cancer vaccine is a therapeutic vaccine thus, treating an existing cancer. In some embodiments, the cancer vaccine is a prophylactic vaccine thus, preventing the development of cancer. In one embodiment, both types of vaccines have the potential to reduce the burden of cancer. In one embodiment, treatment or therapeutic vaccines are administered to cancer patients and are designed to strengthen the body's natural defenses against cancers that have already developed. In one embodiment, therapeutic vaccines may prevent additional growth of existing cancers, prevent the recurrence of treated cancers, or eliminate cancer cells not killed by prior treatments. In some embodiments, prevention or prophylactic vaccines are administered to healthy individuals and are designed to target cancer in individuals who present high risk for the disease. In one embodiment, the cancer vaccine is an antigen/adjuvant vaccine. In one embodiment, the cancer vaccine is a whole cell tumor vaccine. In one embodiment, the cancer vaccine is a dendritic cell vaccine. In one embodiment, the cancer vaccine comprises viral vectors and/or DNA vaccines. In one embodiment, the cancer vaccine is an idiotype vaccine.

[00143] In one embodiment, the compound is administered in combination with an anti-cancer chemotherapeutic agent. In one embodiment, the anti-cancer chemotherapeutic agent is an alkylating agent, such as but not limited to cyclophosphamide. In one embodiment, the anticancer chemotherapeutic agent is a cytotoxic antibiotic such as but not limited to doxorubicin. In one embodiment, the anti-cancer chemotherapeutic agent is an antimetabolite, such as but not limited to methotrexate. In one embodiment, the anti-cancer chemotherapeutic agent is a vinca alkaloid, such as but not limited to vindesine. In some embodiments, the anti-cancer chemotherapeutic agents include platinum compounds such as but not limited to, carboplatin, and taxanes such as docetaxel. In one embodiment, the anti-cancer chemotherapeutic agent is an aromatase inhibitor such as but not limited to anastrazole, exemestane, or letrozole.

[00144] In one embodiment, the compound is administered in combination with a Bax activity modulator such as alisol B acetate. In one embodiment, the compound is administered in combination with an angiotensin II receptor blocker such as losartan. In one embodiment, the compound is administered in combination with selenium, green tea cachecins, saw palmetto, lycopene, vitamin D, dietary soy, genistein or isoflavone.

[00145] In one embodiment, the compound is administered in combination with antineoplastic agents, such as alkylating agents, antibiotics, hormonal antineoplastics and antimetabolites. Examples of useful alkylating agents include alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa and uredepa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophos-phoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol and pipobroman. More such agents will be known to those having skill in the medicinal chemistry and oncology arts.

[00146] In some embodiments, other agents suitable for combination with the compounds of this invention include protein synthesis inhibitors such as abrin, aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride, 5 -fluoro tryptophan, fusidic acid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O-methyl threonine, modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin, ricin, a-sarcin, Shiga toxin, showdomycin, sparsomycin, spectinomycin, streptomycin, tetracycline, thiostrepton and trimethoprim. Inhibitors of DNA synthesis, including alkylating agents such as dimethyl sulfate, mitomycin C, nitrogen and sulfur mustards, MNNG and NMS; intercalating agents such as acridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene, ethidium bromide, propidium diiodide-intertwining, and agents such as distamycin and netropsin, can also be combined with compounds of the present invention in pharmaceutical compositions. DNA base analogs such as acyclovir, adenine, β-1-D-arabinoside, amethopterin, aminopterin, 2- aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil, 2 ' -azido-2 ' -deoxynucliosides, 5-bromodeoxycytidine, cytosine, β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5-fluorouracil, hydroxyurea and 6-mercaptopurine also can be used in combination therapies with the compounds of the invention. Topoisomerase inhibitors, such as coumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors of cell division, including colcemide, colchicine, vinblastine and vincristine; and RNA synthesis inhibitors including actinomycin D, a-amanitine and other fungal amatoxins, cordycepin (3 ' - deoxyadenosine), dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin and streptolydigin also can be combined with the compounds of the invention to provide pharmaceutical compositions.

[00147] In some embodiments, this invention provides for the use of a compound as herein described for treating, reducing the severity of, reducing the incidence of, or reducing pathogenesis of cancer in a subject. In another embodiment, the cancer comprises adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewings family of tumors (Pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic, leukemia, oral cavity cancer, liver cancer, lung cancer, non small cell lung cancer, small cell, lymphoma, AIDS-related lymphoma, central nervous system (primary), lymphoma, cutaneous T-cell, lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metasatic squamous carcinoma, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, exocrine, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, Sezary syndrome, skin cancer, cutaneous T-cell lymphoma, skin cancer, Kaposi's sarcoma, skin cancer, melanoma, small intestine cancer, soft tissue sarcoma, soft tissue sarcoma, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, unusual cancer of childhood, vaginal cancer, vulvar cancer, Wilms' tumor, or any combination thereof.

[00148] It is to be understood, that any cell whereby stimulation of apoptosis by methods disclosed herein is desired, or via the use of any of the active compounds or compositions herein described, is to be considered as part of the present invention. Diseased cells may in turn comprise cells of hematopoietic, neural or mesenchymal origin, including cells of neuronal, cardiac, muscular, connective, hepatic, osteocytic, adipose, thymic, erythroid, myeloid or epithelial origin, or any combination thereof.

[00149] Another mechanism for regulating cellular apoptosis provided for by the present invention is via suppression of the cell death process. Therefore, in another embodiment of the present invention there is provided a method for inhibiting cellular apoptosis, the method comprising the step of contacting a cell an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity.

[00150] This invention encompasses methods of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject which, in some embodiments, comprises the step of administering an agent which diminishes or abrogates mitochondrial Na7Ca²⁺ NCLX exchanger activity.

[00151] In one embodiment, this invention provides for the design of small molecules that stabilize the agent, thereby promoting or enhancing mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. In another embodiment, this invention provides for the design of small molecules that stabilize the agent, thereby diminishing or abrogating mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity. [00152] In one embodiment, molecular replacement may be used for the design of the small molecules herein described to obtain structural information about a crystallized molecule or molecular complex whose structure is unknown, whereby, in one embodiment, it comprises the steps of generating an X-ray diffraction pattern from the crystallized molecule or molecular complex. Structure coordinates may then be applied to the X-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown. Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used in another embodiment, to solve crystal structures that can not be determined directly.

[00153] As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration or electron density map which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include in one embodiment enzymic active sites, or structural subdomains in another embodiment, or epitopes in another embodiment, or functional domains in another embodiment, or signal sequences in another embodiment. A variety of structural formats for the input and output means can be used to input and output the information.

[00154] A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify structural motifs or electron density maps. A skilled artisan can readily recognize that any one of the publicly available computer modeling programs can be used as a search means.

[00155] In one embodiment, inhibition of apoptosis by methods disclosed above may be conducted in cells that are healthy, or in cells that are diseased. It is to be understood, that any diseased cell whereby suppression of apoptosis by methods disclosed herein is desired, or via the use of any of the active compounds or compositions herein described, is to be considered as part of the present invention. Cells may in turn comprise cells of hematopoietic, neural or mesenchymal origin, including cells of neuronal, cardiac, muscular, connective, hepatic, osteocytic, adipose, thymic, erythroid, myeloid or epithelial origin, or any combination thereof.

[00156] In one embodiment, the compounds and compositions of this invention alter the course of, or treat diseases or pathologic conditions, which include, but are not limited to, AIDS, neurodegenerative, cardiovascular, skeletal, inflammatory or infectious or autoimmune diseases or disorders, or any combination thereof.

[00157] For example, the invention provides methods for treating a diseased cell associated with such pathologic conditions such as neurodegenerative diseases. In one embodiment, the neurodegenerative disease or disorder comprises an injury, disease, disorder or condition of the central nervous system (CNS). In another embodiment, the neurodegenerative disease or disorder comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, diabetic neuropathy or amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), Ullrich muscular dystrophy (UMD), Duchenne muscular dystrophy, the encephalopathy associated with acquired immunodeficiency disease (AIDS), glutamate-dependent excitotoxicity in the CNS, or any combination thereof. In another embodiment, the neurodegenerative disease or disorder comprises spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, optic nerve injury, or injury caused by tumor excision, or any combination thereof. In another embodiment, the subject is at risk for a neurodegenerative disease or disorder. Replenishment of nerve cell populations is limited, with diseases resulting in nerve cell death proving an irreversible process. In one embodiment, selective blocking of apoptosis of specific nerve cell populations at risk in a given neurodegenerative disease may provide a means for control of disease progression.

[00158] In one embodiment, the neurodegenerative disease or disorder comprises epilepsy, amnesia, anxiety, hyperalgesia, psychosis, seizures, oxidative stress, opiate tolerance and dependence, a psychosis or psychiatric disorder comprising an anxiety disorder, a mood disorder, schizophrenia or a schizophrenia-related disorder, drug use or dependence or withdrawal, or a memory loss or cognitive disorder, or any combination thereof.

[00159] In another embodiment, neurodegenerative disease or disorder comprises facial nerve (Bell's) palsy, glaucoma, Alper's disease, Batten disease, Cockayne syndrome, Guillain-Barre syndrome, Lewy body disease, Creutzfeld-Jakob disease, or a peripheral neuropathy such as a mononeuropathy or polyneuropathy comprising adrenomeloneuropathy, alcoholic neuropathy, amyloid neuropathy or polyneuropathy, axonal neuropathy, chronic sensory ataxic neuropathy associated with Sjogren's syndrome, diabetic neuropathy, an entrapment neuropathy, nerve compression syndrome, carpal tunnel syndrome, a nerve root compression that may follow cervical or lumbar intervertebral disc herniation, giant axonal neuropathy, hepatic neuropathy, ischemic neuropathy, nutritional polyneuropathy due to vitamin deficiency, malabsorption syndromes or alcoholism, porphyric polyneuropathy, a toxic neuropathy caused by organophosphates, uremic polyneuropathy, a neuropathy associated with a disease or disorder comprising acromegaly, ataxia telangiectasia, Charcot-Marie-Tooth disease, chronic obstructive pulmonary diseases, Fabry's disease, Friedreich ataxia, hypoglycemia, IgG or IgA monoclonal gammopathy (non-malignant or associated with multiple myeloma or with osteosclerotic myeloma), lipoproteinemia, polycythemia vera, Refsum's syndrome, Reye's syndrome or Sjogren-Larrson syndrome, a polyneuropathy associated with various drugs, with hypoglycemia, with infections as HIV infection, with cancer, or any combination thereof. [00160] Infection of the central nervous system, such as, for example, in meningitis, or encephalomyelitis, is often accompanied by an inflammatory response, which is destructive to the tissue. Such scenarios benefit, in one embodiment, by the methods/compositions/kits of this invention.

[00161] In one embodiment, this invention provides for methods of treatment of diseases or disorders involving the central nervous system, including, inter alia, pain, myasthenia gravis (MG), fronto-temporal dementia (FI D), stroke, traumatic brain injury, HIV-associated dementia, encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, cerebral ischemia- induced injury, age-related retinal degeneration, or any combination thereof,

[00162] In another embodiment, this invention provides for methods of treatment of diseases and disorders related to degenerative or atrophic conditions, which may include, but are not limited to, autoimmune diseases and cerebrovascular and neurodegenerative diseases or disorders in the central and peripheral nervous system, or any combination thereof.

[00163] In another embodiment, the invention provides methods for treatment of central nervous system damage as a result of an inflammatory response. The phrase "central nervous system damage" or "CNS damage" refers, in some embodiments, to the result of a disease process or injury that is characterized by destruction of, or harm to, cells of the brain or the spinal cord, such that the normal motor and sensory control function of the brain or spinal cord is disrupted. CNS damage shall be understood to encompass, for example, the result of an acute traumatic break or injury of the spine that completely or partially severs the spinal cord, the result of a stroke, the result of chronic disease such as multiple sclerosis, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and neurodegeneration of aging, and the result of cancerous tumors forming within the central nervous system. A subject suffering from CNS damage is deemed to also be suffering from at least a partial disruption of motor or of sensory function, or of both motor and sensory function as a result of the CNS damage.

[00164] In another embodiment, this invention provides for methods of treatment of diseases and disorders related to abnormally high levels of apoptosis, which may include, but are not limited to, cardiovascular disease. In one embodiment, diseased cells associated with abnormally high levels of apoptosis that may be targeted for apoptotic suppression by the molecules of the present invention comprise, for example, cells associated with cardiovascular disease, wherein suppression of apoptosis in cardiocytes following an ischemic event may provide a means of limiting damage associated with myocardial infarctions. Evidence for the existence of apoptosis in the human heart has been reported in various cardiac diseases, including ischemic and nonischemic heart failure, myocardial infarction and arrhythmias, congestive myocardiopathy, hypertrophic obstructive myocardiopathy, hypertrophic non-obstructive myocardiopathy, idiopathic myocardiopathy, angina pectoris, myocardial infarction, poor prognosis of myocardial infarction, chronic cardiac insufficiency, etc. Examples of angina pectoris include effort angina, exertional angina, rest angina, silent myocardial ischemia, and silent angina; myocardial infarction includes acute myocardial infarction. Among the most potent stimuli that elicit cardiomyocyte apoptosis are oxygen radicals (including NO), cytokines (FAS/TNF alpha; family of cytokines) and growth factors/energy deprivation. Several complex signal transduction pathways have been implicated in execution of cardiomyocyte apoptosis, including Fas/TNF alpha, receptor signaling, stress- or mitogen-activated protein kinases (SAPK/MAPK), sphingolipids metabolites (ceramide), G-protein-coupled receptor (GPCR) signaling (Gc¾i, G«xq) and NF-κΒ activation. Apoptosis of cardiac myocytes may contribute to progressive pump- failure, arrhythmias and cardiac remodeling.

[00165] In another embodiment, cells infected with certain pathogens succumb to high leyels of apoptosis, following infection. Some examples are cells infected with HIV, reovirus, Shigella and Salmonella. Suppression of apoptosis in these cells may alter the course of disease, providing a source of therapy for a myriad of diseases associated with high levels of apoptosis.

[00166] The compositions and compounds of the present invention, in one embodiment, may also be used to diagnose diseases associated with abnormally high levels of apoptosis, which, in one embodiment, may be a neurodegenerative or a cardiovascular disease. In one embodiment, levels of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger could be measured in a tissue and correlated with levels of apoptosis. In another embodiment, the compositions and compounds of the present invention may be used to diagnose diseases associated with low high levels of apoptosis, which, in one embodiment, may be a neoplastic disease.

[00167] By the term "reducing the severity of the pathologic condition", it is to be understood that any reduction via the methods, compounds and compositions disclosed herein, is to be considered encompassed by the invention. Reduction in severity may, in one embodiment comprise enhancement of survival, or in another embodiment, halting disease progression, or in another embodiment, delay in disease progression, or in another embodiment, diminishment of pain, or in another embodiment, delay in disease spread to alternate sites, organs or systems. The identification of modulators of apoptosis thus has enormous clinical significance. It is to be understood that any clinically beneficial effect that arises from the methods, compounds and compositions disclosed herein, is to be considered encompassed by the invention.

[00168] By the term "abnormal level of apoptosis" it is to be understood that increased apoptosis, the presence of apoptosis, decreased apoptosis or the absence of apoptosis, as compared to unafflicted individuals, as a consequence of the pathologic condition, is herein considered, each of which represents an embodiment of the invention. [00169] Compounds/compositions comprising nucleic acid sequences encoding a mitochondrial Na⁺/Ca²⁺ NCLA exchanger, and vectors expressing same, provide an enormous reservoir of therapeutic potential for stimulation of apoptosis in cell populations. As just one application of such, it can be envisioned that the methods of this invention can be useful for increasing or decreasing the level of apoptosis of a population of cells ex vivo, including cells in culture or in an individual. In one embodiment, over-expression of a mitochondrial Na⁺/Ca²⁺ NCLA exchanger may be useful as a method of increasing apoptosis. In another embodiment, expression of a non-functional mitochondrial Na⁺/Ca²⁺ NCLA exchanger may be useful as a method of inhibiting apoptosis.

[00170] In one embodiment, a molecule can be used to treat a mixed population of cells in culture, in order to selectively induce apoptosis in one population, thereby facilitating selection of a remaining population. Such a method may entail screening of molecules identified as having pro-apoptotic ability for their ability to be more readily taken up by desired target cell populations. Such methods are well within the level of skill in the art.

[00171] Screening methods for measuring Ca²⁺ influx comprise, in one embodiment, using Fura-2 and the mitochondrial-targeted ratiometric probe, pericam (Nagai et al. 2001: PNAS 98:3197- 3202). Mitochondrial-free Ca²⁺ concentration may be monitored in cells expressing ratiometric- pericam-mt, using an imaging microscope. Mitochondrial Ca²⁺ concentration then presented as a fluorescence ratio. In another embodiment, a non-invasive ion-selective vibrating probe is used to measure calcium flux, in a cell. This type of vibrating probe is known and is described in several sources, including Smith (Non-invasive Ion Probes— Tools for Measuring Transmembrane Flux, Nature, Dec. 7, 1995) and in more detail in Smith et al. (The Vibrating Ca²⁺ Electrode: A New Technique for Detecting Plasma Membrane Regions of Ca²⁺ Influx and Efflux Methods in Cell Biology 40:115-134 (1994)). A variety of calcium indicators are known in the art, including but not limited to, for example, fura-2 (McCormack et al, 1989 Biochim. Biophys. Acta 973:420); mag-fura-2; ETC (U.S. Pat. No. 5,501,980); fluo-3, fluo-4 and fluo-5N (U.S. Pat. No. 5,049,673); rhod-2; benzofhiaza-^ 1; and benzothiaza-2. For monitoring an indicator of altered mitochondrial function that is a cellular response to elevated intracellular calcium, compounds that induce increased cytoplasmic and mitochondrial concentrations of Ca ⁺, including calcium ionophores, are well known to those of ordinary skill in the art, as are methods for measuring intracellular calcium and intramitochondrial calcium (see, e.g., Gunter and Gunter, 5 1994 J. Bioenerg. Biomembr. 26: 471; Gunter et al, 1998 Biochim. Biophys. Acta 1366:5; McCormack et al, 1989 Biochim. Biophys. Acta 973:420; Orrenius and Nicotera, 1994 Neural. Transm. Suppl. 43:1; Leist and Nicotera, 1998 Rev. Physiol. Biochem. Pharmacol. 132:79; and 10 Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals— Sixth Ed., Molecular Probes, Eugene, Oreg.). Accordingly, a person skilled in the art may readily select a suitable ionophore (or another compound that results in increased cytoplasmic and/or mitochondrial concentrations of Ca²⁺) and an appropriate means for detecting intracellular and/or intramitochondrial calcium for use in the present invention, according to the instant disclosure and to well known methods.

[00172] Screening methods for assessing potential stimulators/accelerators or down- regulators/suppressors of apoptosis comprise the step of contacting a cell with a potential compound and measuring apoptosis in the cell. In one embodiment, measuring comprises imaging of individual cells, a group of cells, a tissue, an organ or a combination thereof, and may, in one embodiment, be accomplished with computed tomography, computed radiography, magnetic resonance imaging, fluorescence microscopy, angiography, arteriography, or a combination thereof. In one embodiment, a cell is contacted with a nucleic acid of this invention, ex vivo, and is subsequently implanted in a subject. In one embodiment, the cell is, inter alia, labeled with a labeling agent as described herein, and may further comprise a therapeutic compound, and/or in another embodiment, the therapeutic compound is labeled with a labeling agent, and in one embodiment, the delivery of the cell and/or therapeutic compound may be verified by imaging the labeling agent.

[00173] In one embodiment, the imaging methods are conducted on a subject. In another embodiment, the imaging methods are conducted on a sample taken from a subject.

[00174] In one embodiment, the imaging methods as described herein may comprise near infrared fluorescence imaging. In one embodiment, an advantages of such optical imaging methods may include the use of non-ionizing low energy radiation, high sensitivity with the possibility of detecting micron-sized objects, continuous data acquisition, and the development of potentially cost-effective equipment. Optical imaging can be carried out at different resolutions and depth penetrations. Fluorescence-mediated tomography (FMT) can three-dimensionally localize and quantify fluorescent probes in deep tissues at high sensitivity. Several NIR fluorochromes have recently been coupled to affinity molecules (Becker A. et al. Nature Biotechnology, 19:327-331, 2001; Folli S. et al. Cancer Research, 54:2643-2649, 1994), and can be adapted to comprise the nucleic acids of this invention, as will be appreciated by one skilled in the art.

[00175] In one embodiment, imaging methods may comprise nuclear imaging methods, such as single photon emission methods (such as planar scintigraphy and tomography, or SPECT), and Positron Emission Tomography (PET). Resolution of clinical scanners may be limited to about 5-6 mm for PET and ~1 cm for SPECT, thus, nuclear imaging methods are often used to complement the information provided by CT and/or MRI scans in the context of multimodality imaging. In one embodiment, nuclear imaging is used in particular because of its sensitivity to extremely small quantities of matter. For example, it has recently been estimated that PET can detect as few as a cluster of 250 cells each bearing 30 Bq of ¹⁸F, which corresponds to 2.1 fg.

[00176] In another embodiment, different iodine isotopes can be chosen for radioactive labeling of compounds. In one embodiment, '"I, ^l"l and ^1J1I can be used to obtain molecules with the same chemical and biological characteristics but different imaging and dosimetric properties. In one embodiment, the isotope for imaging is ¹²³I (159 keV), or in another embodiment, 37 MBq of ¹²³I-MIBG, which results in an exposure to a radiation dose no higher than 1.8 MBq of ^I31I- MIBG.

[00177] In radioimmunotherapy (RIT), cytotoxic radiation from therapeutic radioisotopes is delivered to tumors via antibodies or peptides that bind to tumor-specific or tumor-associated antigens. Radioactive metal ions can be attached to an antibody through a metal chelating agent. One advantage for RIT over other immunotherapies, such as immunotoxins, is that there is no need to target every tumor cell to cause an antitumor effect at the cellular level because nontargeted cells can be irradiated and often killed by radiation from targeted neighboring cells. With immunotoxins, each tumor cell must be targeted for the antitumor effect to occur at the cellular level.

[00178] In another embodiment, some of the radioisotopes may serve a dual purpose, such as, in one embodiment, for imaging the sites to which the radioisotope is delivered, and in another embodiment, as part of radiotherapy, including radioimmunotherapy. In one embodiment, ¹³¹I and Y are used. ¹³¹L in one embodiment, may be attached to an antibody or peptide by simple techniques (such as the IODOGEN or chloramine-T methods), and may be imaged by instrumentation which detects γ-emission, while β-emission serves for therapeutic application in the subject.

[00179] It is to be understood that any assay for measuring a particular activity which is modulated by the therapeutic compound may be employed, as a means of determining the efficacy of the compound, in one embodiment, optimal loading of the compound, in another embodiment, timing and dosage, in another embodiment, or a combination thereof.

[00180] Any number of cells or cell lines may be incubated with tagged molecules and targeting of desired cells and/or uptake may be demonstrated by conventional means, including microscopy, FACS analysis, western blot analysis, and others.

[00181] Imaging methods include in vivo imaging, MR-imaging or NIRF analysis, as well as fluorescence microscopy of excised target tissue, the images of which may be compared to those obtained by MIR or NIRF.

[00182] Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[00183] Further, in another embodiment, the pharmaceutical compositions are administered as a suppository, for example a rectal suppository or a urethral suppository. Further, in another embodiment, the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In a further embodiment, the pellet provides for controlled release of an agent over a period of time. In yet another embodiment, the pharmaceutical compositions are administered in the form of a capsule.

[00184] The compositions as set forth herein may be in a form suitable for intracranial administration. In some embodiments, direct methods to introduce therapeutic agents into the brain substance include the use of devices and needles, such as in the case of intrathecal and intracerebroventricular delivery. In other embodiments, direct methods to introduce therapeutic agents into the brain substance include the use of magnets coupled to the composition of the invention for site-directed delivery. In other embodiments, direct methods to introduce therapeutic agents into the brain substance include the use of heat-activated compounds coupled to the composition of the invention for site-directed delivery. In one embodiment, delivery of the agent to the CNS is a function of its ability to access a relevant target site within the CNS.

[00185] The compositions as set forth herein may be in a form suitable for intransal administration. In some embodiments, intranasal delivery insures CNS delivery, upon crossing the olfactory nerves, the trigeminal nerves, or both. Intranasal delivery does not require any modification of the therapeutic agents and does not require that drugs be coupled with any carrier like in the case of drug delivery across the BBB. The olfactory neural pathway provides two pathways across the BBB. The intraneuronal pathway involves axonal transport and requires hours to days for drugs to reach different brain regions, while an extraneuronal pathway into the brain relies on bulk flow transport through perineural channels, which deliver drugs directly to the brain parenchymal tissue and/or CSF, and allows therapeutic agents to reach the CNS within minutes. In some embodiments, intranasal delivery is via the intraneuronal pathway. In other embodiments, intranasal delivery is via the extraneuronal pathway. In another embodiment, intranasal delivery is via a combination of the intraneuronal and extraneuronal pathways.

[00186] For intranasal administration or application by inhalation, solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier suitable. Such an aerosol may comprise any agent described herein.

[00187] In one embodiment, the route of administration may be parenteral, or a combination thereof. In another embodiment, the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation (aerosol), nasal aspiration (spray), intranasal (drops), sublingual, oral, aerosol or suppository or a combination thereof. In one embodiment, the dosage regimen will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, body weight, and response of the individual patient, etc.

[00188] For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas. Ampoules are convenient unit dosages. Such a suppository may comprise any agent described herein.

[00189] Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the new compounds and use the lyophilisates obtained, for example, for the preparation of products for injection.

[00190] For liquid formulations, pharmaceutically acceptable carriers may be aqueous or nonaqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[00191] In one embodiment, compositions of this invention are pharmaceutically acceptable. In one embodiment, the term "pharmaceutically acceptable" refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound for use in the present invention. This term refers to the use of buffered formulations as well, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration.

[00192] In one embodiment, a composition of or used in the methods of this invention may be administered alone or within a composition. In another embodiment, compositions of this invention admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not delete riously react with the active compounds may be used. In one embodiment, suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. In another embodiment, the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. In another embodiment, they can also be combined where desired with other active agents, e.g., vitamins.

[00193] In one embodiment, the therapeutic compositions of the present invention may comprise the composition of this invention and one or more additional compounds effective in preventing or treating neurodegenerative conditions. In some embodiments, the additional compound may comprise an immunomodulating compound.

[00194] In one embodiment, the immunomodulating agent is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. In one embodiment, the non-steroidal anti-inflammatory agent is a cox-1 inhibitor. In one embodiment, the non-steroidal anti-inflammatory agent is a cox-2 inhibitor. In one embodiment, the nonsteroidal anti-inflammatory agent is a cox-1 and cox-2 inhibitor. In some embodiments, non- steroidal anti-inflammatory agents include but are not limited to aspirin, salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, or celecoxib. In one embodiment, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In one embodiment, the steroidal anti-inflammatory agent is a corticosteroid.

[00195] Dosing is dependent on the cellular responsiveness to the administered molecules/compounds or compositions comprising same. In general, the doses utilized for the above described purposes will vary, but will be in an effective amount to exert the desired effect, as determined by a clinician of skill in the art. As used herein, the term "pharmaceutically effective amount" refers to an amount of a compound as described herein, which will produce the desired alleviation in symptoms or other desired phenotype in a patient.

[00196] In one embodiment of the invention, the concentrations of the compounds will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration and the individual tolerability of the compositions.

[00197] In some embodiments, any of the compositions of this invention will comprise a compound, in any form or embodiment as described herein. In some embodiments, any of the compositions of this invention will consist of a compound, in any form or embodiment as described herein. In some embodiments, any of the compositions of this invention will consist essentially of a compound, in any form or embodiment as described herein. In some embodiments, the term "comprise" refers to the inclusion of the indicated active agent, such as the compound of this invention, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of" refers to a composition whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In some embodiments, the term "consisting essentially of" may refer to components which facilitate the release of the active ingredient. In some embodiments, the term "consisting" refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient.

[00198] It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular conditions and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.

[00199] In one embodiment, the compounds of the invention may be administered acutely for acute treatment of temporary conditions, or may be administered chronically, especially in the case of progressive, recurrent, or degenerative disease. In one embodiment, one or more compounds of the invention may be administered simultaneously, or in another embodiment, they may be administered in a staggered fashion. In one embodiment, the staggered fashion may be dictated by the stage or phase of the disease.

[00200] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. [00201] In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[00202] In one embodiment, the pharmaceutical compositions provided herein are controlled- release compositions, i.e. compositions in which the anti-estrogen compound is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which all of the compound is released immediately after administration. In one embodiment, the controlled- or sustained-release compositions of the invention are administered as a single dose. In another embodiment, compositions of the invention are administered as multiple doses, over a varying time period of minutes, hours, days, weeks, months or more. In another embodiment, compositions of the invention are administered during periods of acute disease. In another embodiment, compositions of the invention are administered during periods of chronic disease. In another embodiment, compositions of the invention are administered during periods of remission. In another embodiment, compositions of the invention are administered prior to development of gross symptoms.

[00203] In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose. In another embodiment, the controlled-release system may be any controlled release system known in the art.

[00204] The compositions may also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[00205] Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[00206] Also comprehended by the invention are compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. The modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds. Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer- compound abducts less frequently or in lower doses than with the unmodified compound.

[00207] The preparation of pharmaceutical compositions that contain an active component, for example by mixing, granulating, or tablet-forming processes, is well understood in the art. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the compound is mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. For parenteral administration, the compound is converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other substances.

[00208] An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimefhylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[00209] For use in medicine, the salts are pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts, which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[00210] As defined herein, the term "contacting" means that the compound of the present invention is introduced into a subject receiving treatment, and the compound is allowed to come in contact with the cells in vivo.

[00211] As used herein, the term "treating" includes preventive as well as disorder remittive treatment. As used herein, the terms "reducing", "suppressing" and "inhibiting" have their commonly understood meaning of lessening or decreasing. As used herein, the term "progression" means increasing in scope or severity, advancing, growing or becoming worse. As used herein, the term "recurrence" means the return of a disease after a remission.

[00212] As used herein, the term "administering" refers to bringing a subject in contact with a compound of the present invention. As used herein, administration can be accomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example humans. In one embodiment, the present invention encompasses administering the compounds of the present invention to a subject.

[00213] Although the pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical composition suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with little, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and other mammals. [00214] In one embodiment, "preventing, or treating" refers to any one or more of the following: delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics. In one embodiment, "treating" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove.

[00215] In another embodiment, "symptoms" may be any manifestation of a disease or pathological condition as described hereinabove.

[00216] In some embodiments, any of the compositions or sequences of and for use in the methods of the present invention will comprise one or more isolated nucleic acids or polypeptides encoding one or more Na⁺/Ca²⁺ NCLX exchangers or microRNAs described herein, in any form or embodiment as described herein. In some embodiments, any one or more of the compositions or sequences of and for use in the methods of the present invention will consist of one or more isolated nucleic acids or polypeptides encoding one or more Na⁺/Ca²⁺ NCLX exchangers or microRNAs, in any form or embodiment as described herein. In some embodiments, any of the compositions or sequences of and for use in the methods of the present invention will consist essentially of one or more isolated nucleic acids or polypeptides encoding one or more Na⁺/Ca²⁺ NCLX exchangers or microRNAs, in any form or embodiment as described herein. In some embodiments, the term "comprise" refers to the inclusion of other nucleic or amino acid sequences, such as promoters, enhancers, etc. that may be known in the art. In some embodiments, the term "consisting essentially of refers to a composition or sequence, which has the specific nucleic acids or polypeptides encoding one or more Na⁺/Ca²⁺ NCLX exchangers or microRNAs. However, other compositions or sequences may be included that are not involved directly in the utility of the Na⁺/Ca²⁺ NCLX exchangers. In some embodiments, the term "consisting" refers to a composition or sequence having a particular nucleic acids or polypeptides encoding one or more Na⁺/Ca²⁺ NCLX exchangers or microRNAs of the present invention, in any form or embodiment as described herein.

[00217] In one embodiment, methods of the present invention involve treating a subject by, inter alia, controlling the expression, production, and activity of cytokines, chemokines and interleukins; anti-oxidant therapy; anti-endotoxin therapy or any combination thereof. [00218] The administration mode of the compounds and compositions of the present invention, timing of administration and dosage, i.e. the treatment regimen, will depend on the type and severity of the injury, disease or disorder, and the age and condition of the subject. In one embodiment, the compounds and compositions may be administered concomitantly. In another embodiment, the compounds and compositions may be administered at time intervals of seconds, minutes, hours, days, weeks or more.

[00219] In one embodiment, the method comprises administering the agents in a composition in a form suitable for administration via an intracranial route. In another embodiment, the composition is in a form suitable for administration via an intranasal route. In another embodiment, the method comprises administering the composition via an oral, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical route.

[00220] It is to be understood that any method of this invention, as herein described, encompasses the administration of a compound as herein described, or a composition comprising the same, to the subject, in order to treat the indicated disease, disorder or condition. The methods as herein described each and/or all may further comprise administration of an additional therapeutic agent as herein described, and as will be appreciated by one skilled in the art.

[00221] The following examples are intended to illustrate but not limit the present invention.

EXAMPLES MATERIALS AND EXPERIMENTAL METHODS [00222] Cell culture and transfection. CHO, HEK293-T and primary rat cortical neurons were cultured were cultured in DMEM and plated on glass cover-slips prior to transfection procedures. Primary rat cortical neurons were cultured and transfection of HEK293-T cells was performed using CaP0₄ precipitation. CHO cells were transfected using Lipofectamine 2000 (Invitrogene) or Transfect-it CHO reagent (Mirus) according to the manufacturer's protocol. Fluorescent ion measurements and cell harvesting were conducted 48-72 h after transfection.

[00223] Plasmid and siRNA preparation. Preparation of the human NCLX plasmid was described previously. To generate NCLXS468T plasmid we replaced a Notl fragment from the a.2 S273T plasmid with the corresponding fragment of the NCLX plasmid. The mouse NCLX variant plasmid referred as NCLX L-Mu was generously provided by Dr. Lytton. Double stranded siRNAs used for silencing NCLX were obtained from Ambion. The sequence of 21 nucleotides corresponding to the sense strands used for the NCLX siRNA was AACGGCCACUCAACUGUCUtt (SEQ NO: 6) and for the control siRNA was AACGCGCAUCCAACUGUCUtt (SEQ NO: 7).

[00224] Fluorescent measurements of Ca²⁺ signals. The imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany). Fluorescent imaging measurements were acquired with Imaging Workbench 4.0 (Axon Instruments, Foster City, CA). Cytosolic Ca²⁺ was measured using Fura-2 AM loaded cells, excited at 340- and 380nm wavelength light and imaged with a 510nm longpass filter. The ratiometric fluorescent data obtained from individual experiments was normalized to percentage of the starting value in order to average different independent experiments (). Mitochondrial Ca²⁺ was monitored in cells transiently expressing the mitochondrial targeted ratiometric-pericam (RP-mt). RP-mt excitation was at 430 nm and emission monitored at 535 nm bandpass filter. Mitochondrial Ca²⁺ levels are presented as I-F430 (F/F₀).

[00225] Ringer's solutions contained 130 mM NaCl, 20 mM Hepes, 15 mM glucose, 5 mM KCl, 0.8 mM MgCl and pH adjusted to 7.4. Ringer's solution was supplemented with 2 mM CaCl and 40 μΜ ATP in the presence or absence CGP-37157 (10 μΜ) as indicated. Measurements of mitochondrial Ca²⁺ efflux in digitonin-permeabilized (0.007% digitonin) cells were performed in sucrose buffer containing 250 mM Sucrose, 10 mM Hepes, 5 mM succinate, 2.5 mM KH₂P0₄, 0.4 mM EGTA, 1 μΜ ruthenium red and pH was adjusted to 7.4 with KOH.

[00226] Confocal microscopy. Primary rat cortical neurons grown on glass coverslips and fixed with 4% paraformaldehyde in PBS solution for 15 minutes and washed three times with PBS (pH 7.4) (24). Fixed cells were blocked with normal horse serum containing 0.1% tween and then incubated overnight at 4-8° C with the NCLX antibody (1:100). Cells were washed three times with PBS and incubated with Cy2 conjugated anti-rabbit IgG for one hour at room temperature. After rinsing with PBS, coverslips were mounted with Immu-mount (Shandon, USA) and images were acquired using a Zeiss LSM510 confocal microscope.

[00227] Immunoelectron microscopy. Immunohistochemistry for NCLX was carried out using the avidin-biotin method (Vectastain). Briefly, young, 30 day old Sprague-Dawley rats (n=4) were perfused intracardially with 2.5% paraformaldehyde and 0.1% gluteraldehyde in 0.5M PBS (pH 7.4). Brains were removed and left overnight in the same fixative at 4°C. Each brain was sectioned on a vibrating microtome and 50 μπι sections containing the hippocaampus were incubated for 10 min. in 3% H₂0₂ in 10% methanol to block endogenous peroxidase. After extensive rinses in 0.5 M PBS, the sections were incubated in a blocking solution containing 1% BSA, 4% normal serum (produced in the species in which the secondary antiserum was made) and 0.1% triton-X for 30 min. Incubation overnight in the primary antiserum followed immediately, with 5% of the BSA-containing solution left to mix with the primary AB. Sections were then incubated for 30 min. in biotinylated anti-rabbit IgG (1:200), followed by incubation in Vectastain ABC reagent (Vector Laboratories, Burlington, VT) diluted to 1:100 for 30 min. The HRP was developed in 0.04% diaminobenzidine tetrahydrochloride (DAB) prepared in Tris- imidazole buffer with 0.003% H2O2.

[00228] The immunolabeled tissue was subsequently postfixed with 1% OSO₄ for 30 min, stained en bloc with 1% uranyl acetate for 60 min and dehydrated in ethanols to propylene oxide before embedding flat in epon-araldite between glass slides. Ultrathin (80 nm) sections were cut on a Reichert-Jung ultramicrotome and mounted on copper grids for analysis at 60 kV in a JEOL 100SX transmission electron microscope. Negatives were developed and scanned into Adobe Photoshop for contrast/brightness enhancement and resizing.

[00229] Cell fractionation and immunoblot analysis. Isolation of ER, crude and pure mitochondrial fractions from rat brain and liver was performed. Cell fractionation was performed with minor modifications. Briefly, transfected cells were homogenized to produce total protein sample, Total protein samples were centrifuged at 1500g for 5 min in 4°C. Supernatant was collected and samples were washed with fresh homogenization buffer and re-centrifuged. The pellet, containing mostly plasma membranes, was recovered. Supernatants were collected and centrifuged for 10 min at 12000g (4°C). Supernatant containing ER fragments and cytosolic proteins was saved. The pellet, containing the mitochondria, was then washed three times. All samples were analyzed for protein concentration using the Bradford based method (Bio-Rad). Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblot analysis was carried out using the NCLX-antiserum (or the pre- immune serum) at a 1/2000 dilution, antibodies against β-actin or VDAC1.

RESULTS EXAMPLE 1

Cellular and subcellular localization of NCLX

[00230] The cellular localization of NCLX was determined by western blot analysis of NCLX expression in cellular fractions purified from rat liver (Fig. la) or brain (Fig. lb). NCLX expression was predominantly found in the mitochondrial fraction (Fig. lb). The mouse isoform of NCLX (NCZJL_L-Mu) was then heterologously expressed in HEK293-T cells and the expression of NCLX studied using immunoblot analysis of preparations of plasma membrane, mitochondrial or ER fractions. Expression of NCLX was most dominant in the mitochondrial fraction (Fig. lc). VDAC labeling was performed as a marker of the mitochondrial fractions (lower panel). Mouse cortical neurons were then stained with an antibody for NCLX and colocalization analysis of primary cultured rat cortical neurons was performed for NCLX expression (e and h (green)) costained with mitotracker (d and g (red)) at x40 (d-f) and xlOO (g- i). Projection of images using mitotracker red and NCLX staining (f and i (bottom panel)) showed a large overlap between the fluorescence of NCLX and mitotracker red, indicating co- localization of NCLX to the internal mitochondrial membrane. (Fig. Id and e). Finally, immunoelectron microscopy analysis of the cortical region of rat brain was performed, and indicated that NCLX is present in -mitochondria. NCLX staining was observed in the inner mitochondrial compartments, while in slices treated with the pre-immune serum the mitochondrial membrane staining was not apparent (Fig. lf-g). Rat cortical slices were stained with NCLX antibody (j), or NCLX preimmune serum (k) as described above and imaged by transmission electron microscopy. Black arrows point to NCLX-immunopositive mitochondria (NCLX) and unlabeled mitochondria (Control), respectively. Positive immunolabeling is defined as the presence of black, HRP -precipitate. The scale bar = 0.25 μηι.

EXAMPLE 2

Expression of NCLX enhances mitochondrial Ca²* efflux.

[00231] To determine if NCLX participates in mitochondrial Ca²⁺ transport and homeostasis, changes in cytosolic and mitochondrial [Ca²⁺] were monitored using Fura-2 and the mitochondrial-targeted ratiometric probe, pericam (RP-mt), respectively. For these experiments, HEK293-T cells heterologously expressing the mouse NCLX isoform (A Jf_L-Mu) were employed because of the specific mitochondrial localization of this isoform, which minimizes the contribution of plasma membrane NCLX activity. Cells transfected with NCLX_h-Mu or vector (control) were cotransfected with RP-mt and subsequently superfused with ATP (40 μΜ, at the indicated time) while monitoring mitochondrial Ca²⁺ fluorescence. As shown in Fig. 2a, application of ATP (40 μΜ) was followed by a rise in mitochondrial Ca²⁺ that was two-fold smaller in the NCLX_L-Mu expressing cells, suggesting that mitochondrial Ca²⁺ accumulation is attenuated in the presence of the exchanger. Despite the lower uptake of mitochondrial Ca²⁺, the subsequent rate of mitochondrial Ca²⁺ efflux was -three-fold higher in NCL _L-Mu-expressing cells than in control cells. Differences in uptake and efflux between NCLX_L Mu over- expressing cells and controls were abolished by repeating the same ATP challenge in the presence of CGP-37157, an inhibitor of the mitochondrial Na⁺-dependent Ca ⁺ efflux (Fig. 2a). This indicates that NCLX expression results in lower Ca²⁺ accumulation due to enhanced, CGP- 37157-sensitive, Ca²⁺ efflux.

[00232] To determine whether NCLX mediates capacitative Ca²⁺ entry, cytosolic Ca²⁺ dynamics was examined in NCLX_L-Mu (or vector)-expressing cells loaded with the Ca²⁺ sensitive probe, fura-2. In this set of experiments, capacitative Ca²⁺ entry was activated by sustained Ca²⁺ release from the ER triggered by prolonged application of ATP (40 μΜ) followed by reintroduction of extracellular Ca²⁺, and reflected the trans-mitochondrial flux. The averaged rates of mitochondrial Ca²⁺ accumulation (b) or efflux (c) in the presence or absence of the mitochondrial exchanger inhibitor CGP-37157 are presented. Rates of Ca²⁺, rise triggered by the reapplication of Ca²⁺, in NCLX_L-Mu expressing HEK293-T cells were three-fold higher than in control cells (Fig. 2b). When the experiments were repeated in the presence of CGP-37157, the rate of cytoplasmic Ca²⁺ rise and the decrease in mitochondrial Ca²⁺ were similar in NCLX_L-M - transfected HEK293-T cells and control cells (Fig. 2c-d). This protocol was repeated in the presence or absence of 10 μΜ CGP-37157 (the same traces from Fig. 2d are presented for comparison) in cells expressing control (e) or NCLX_L-Mu (f). The second phase of Ca²⁺ entry is shown. Expression of NCLX_L-Mu was followed by enhanced rise of cytoplasmic Ca²⁺ that was blocked by CGP-37157. Averaged rates of capacitative Ca²⁺ entry monitored in the indicated experiments. * - p< 0.05 (Fig. 2g). To ascertain that the capacitative Ca²⁺ entry is not modulated by NCLX_L-Mu, Fura-2 quenching following Mn²⁺ permeation via this channel was monitored. Similar rates of Mn²⁺ influx were monitored in NCLX_L-M - and vector-expressing cells.

EXAMPLE 3

NCLX knockdown by siRNA results in decreased mitochondrial Ca²⁺ efflux.

[00233] To further investigate whether the activity of the NCLX is largely responsible for the described Na⁺-dependent mobilization of mitochondrial Ca²⁺, mitochondrial Ca²⁺ dynamics was assessed in CHO (Chinese Hamster Ovary) cells in which the activity of endogenously expressed NCLX was silenced. CHO cells were transfected with an siRNA construct aimed to silence NCLX expression or with a 4-base scrambled siRNA of the same construct (control). NCLX or β-actin expression was assessed by western blot analysis in CHO cells transfected with NCLX- or control siRNA and resulted in about 40% decrease in NCLX expression (Fig. 3a). As observed for the RP-mt fluorescence, -30% of the cells were transfected and only these cells were analyzed for the Ca²⁺ measurements, suggesting that silencing in these cells was much higher (~90%). The same experimental protocol as in Fig. 3b was then applied to study mitochondrial Ca²⁺ transport in the NCLX siRNA transfected cells using the RP-mt that was also used as a marker for identifying transfected cells. Rates of mitochondrial Ca²⁺ influx were increased by -50%, while Ca²⁺ efflux rate was totally inhibited, in the NCLX siRNA transfected cells compared to control cells (Fig. 3c). Capacitative Ca²⁺ entry was triggered, and trans- mitochondrial Ca²⁺ efflux rate was monitored in CHO NCLX silenced and control cells. Results exhibit a 2.5 -fold decrease in the apparent trans-mitochondrial Ca²⁺ efflux rate in NCLX silenced cells compared to control cells. The averaged rates of mitochondrial Ca²⁺ accumulation (d) or efflux (e) are shown. Silencing of endogenous NCLX reduced the trans-mitochondrial flux (Fig. 3f). The same experimental paradigm described in Fig. 2d was used for CHO cells expressing control or NCLX siRNA. Averaged rates of capacitative Ca²⁺ entry measured in these experiments is shown in Fig. 3g.

EXAMPLE 4

Mitochondrial NCLX mediates Na⁺- or Li⁺-dependent Ca²* efflux that is inhibited by siRNA silencing or mutagenesis.

[00234] To determine if NCLX mediates Na⁺-dependent Ca²⁺ exchange in mitochondria and to characterize the distinct transport properties of this mitochondrial transporter, mitochondrial Ca²⁺ was monitored in HEK293-T cells co-transfected with RP-mt and the human variant of NCLX, NCLXS468T or pCDNA3.1 (control) plasmids (Fig. 4a). Cells were permeabilized using digitonin (0.007%), and mitochondrial Ca²⁺ was monitored while replacing Na⁺-free with Na⁺- containing solution. Averaged rates of Ca²⁺ efflux in cells expressing either NCLX, NCLXS468T or control are shown in Fig. 4b. Knockdown of NCLX using siRNA decreased mitochondrial Na⁺- (c) or Li⁺- (e) dependent Ca²⁺ efflux activity. Mitochondrial Ca²⁺ was recorded in CHO cells co-transfected with NCLX or control siRNA together with RP-mt plasmid and experiments were performed using the protocol described in (a). Averaged initial rates of mitochondrial Ca²⁺ efflux measured in cells transfected with the NCLX siRNA (Fig. 4d and f) or control (Fig. 4c and e), respectively.

EXAMPLE 5

Increased expression of mitochondrial NCLX as a novel anti-cancer agent

[00235] Subjects suffering from a neoplastic disease are administered a liposome formulation comprising a vector comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger. NCLX expression is assessed by western blot analysis. Tumor size and spread are monitored by MRI and CT scans. Tumor regression is evident.

EXAMPLE 6

NCLX knockdown in treating neurodegenerative conditions

[00236] Subjects suffering from a neurodegenerative condition are administered an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger. NCLX expression is assessed by western blot analysis. The extent and rate of neurodegeneration is examined by MRI and CT scan. Subjects show reduced rates of neurodegeneration.

EXAMPLE 7

Effect of NCLX inhibitor on blood pressure in rats

[00237] Hypertensive rats were infused with CPG 37157, an inhibitor of the mitochondrial Na⁺/Ca ⁺ NCLX exchanger, and their systolic blood pressure was measured over time. CGP 37157 reduces systolic blood pressure over time in treated rats relative to control rats who were not administered CGP 37157 (Figures 5 and 6). Figure 5 shows a comparison of blood pressure measurements in control rats versus rats treated with CPG 37157. Figure 6 presents the data as average blood pressure of the control and experimental groups. In addition, other inhibitors of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger, such as antibodies to a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, siRNA and small molecules which inhibit the function of the exchanger, are administered to hypertensive rats and their systolic blood pressure is measured over time.

Claims

WHAT IS CLAIMED IS:

1. Use of an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in the treatment of a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

2. The use of claim 1, wherein said mitochondrial Na⁺/Ca²⁺ NCLX exchanger is encoded by a nucleic acid sequence shares at least 95% homology with SEQ ID NOs: 1-3.

3. The use of claim 1, wherein said agent is a microRNA comprising a sequence complementary to a fragment of SEQ NOs: 1-3.

4. The use of claim 3, wherein said microRNA has a sequence sharing at least 95% homology with SEQ NO: 6

5. The use of claim 1, wherein said disease is hypertension, metabolic syndrome or a neurodegenerative disease.

6. The use of claim 1, wherein said disease is cardiovascular disease, a skeletal disease, an inflammatory disease, or an infectious disease.

7. The use of claim 6, wherein said disease is AIDS.

8. The use of claim 1, wherein said agent is an antibody specifically recognizing a polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said polypeptide has a sequence sharing at least 95% homology with SEQ NOs: 8-10

9. A method of treating a disease, which is improved by diminishing or abrogating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby treating said disease.

10. An isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said nucleic acid sequence shares at least 95% homology with SEQ ID NOs: 1-3.

11. The isolated nucleic acid of claim 10, wherein expression of said nucleic acid is under the control of a tissue-specific promoter.

12. A vector comprising the nucleic acid of claim 10.

13. The vector of claim 12, wherein said vector comprises a promoter which is regulatable.

14. The vector of claim 12, wherein said vector is a viral vector.

15. The vector of claim 12, wherein said vector is an integrating vector.

16. A polypeptide encoded by the nucleic acid of claim 10.

17. An isolated polypeptide having mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, wherein said isolated polypeptide has a sequence sharing at least 95% homology with SEQ NOs: 8-10.

18. An antibody specifically recognizing the polypeptide of claim 17.

19. The antibody of claim 18, wherein said antibody abrogates or diminishes mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity.

20. A microRNA comprising a sequence complementary to a fragment of SEQ NOs: 1-3.

21. The microRNA of claim 20, wherein said microRNA has a sequence sharing at least 95% homology with SEQ NO: 6.

22. An isolated nucleic acid comprising a nucleic acid sequence encoding a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 13.

23. The isolated nucleic acid of claim 22, wherein expression of said nucleic acid is under control of a tissue-specific promoter.

24. A vector comprising the nucleic acid of claim 22.

25. The vector of claim 24, wherein said vector comprises a promoter which is regulatable.

26. The vector of claim 24, wherein said vector is a viral vector.

27. The vector of claim 24, wherein said vector is an integrating vector.

28. A polypeptide encoded by the nucleic acid of claim 221.

29. An isolated polypeptide comprising an amino acid sequence of a non-functional mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 14.

30. A microRNA comprising a sequence complementary to a fragment of SEQ NO: 13.

31. Use of an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, in a method of inhibiting cellular apoptosis, wherein said agent is:

• an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 13 of null mutants;

• a microRNA having a sequence sharing at least 95% homology with SEQ NO: 6; or

• an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

32. A method of inhibiting cellular apoptosis comprising the step of contacting a cell with an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby inhibiting apoptosis.

33. The method of claim 32, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 13 of null mutants or a microRNA having a sequence sharing at least 95% homology with SEQ NO: 6, wherein said contacting diminishes or abrogates expression, activity, function, or a combination thereof, of said exchanger.

34. The method of claim 32, wherein said agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

35. The method of claim 32, wherein said cell is a cell of the peripheral nervous system (PNS), central nervous system (CNS), immune cell, cardiac cell, or any combination thereof.

36. Use of an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of stimulating or accelerating apoptosis, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3.

37. A method of stimulating or accelerating apoptosis comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby stimulating or accelerating apoptosis.

38. The method of claim 37, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3, wherein said contacting up- regulates expression, activity, function, or a combination thereof, of said exchanger.

39. The method of claim 37, wherein said cell is a preneoplastic cell, a neoplastic cell, an inflammatory cell, an infected cell, or any combination thereof.

40. Use of an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity in a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, wherein said agent is:

• a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 13;

• a microRNA sequence comprising a sequence complementary to a fragment of SEQ ID NOs: 1-3; or

41. A method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which diminishes or abrogates mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition.

42. The method of claim 41, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NO: 13 or a microRNA sequence comprising a sequence complementary to a fragment of SEQ ID NOs: 1-3, wherein said contacting diminishes or abrogates expression of said exchanger.

43. The method of claim 42, wherein said microRNA has a sequence sharing at least 95% homology with SEQ NO: 6.

44. The method of claim 41, wherein said agent is an antibody, which interferes with the function of the mitochondrial Na⁺/Ca²⁺ NCLX exchanger.

45. The method of claim 41, wherein said pathologic condition is a result of AIDS, or a cardiovascular, neurodegenerative, skeletal, inflammatory or infectious disease or disorder, or any combination thereof.

46. Use of an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity in a method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, wherein said agent is a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3.

47. A method of reducing the severity of a pathologic condition associated with abnormal levels of apoptosis in a subject, comprising the step of administering an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger activity, thereby reducing the severity of a pathologic condition.

48. The method of claim 47, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3, and wherein said contacting stimulates or increases expression of said exchanger.

49. The method of claim 47, wherein said pathologic condition is a result of a preneoplastic, neoplastic, autoimmune, or reactive cell disorder, or any combination thereof.

50. Use of an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof in a method of treating a disease, which is improved by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca ⁺ NCLX exchanger, wherein said agent is a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3.

51. A method of treating a disease, which is improved by stimulating or accelerating calcium flux via a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, said method comprising the step of contacting a cell with an agent which stimulates or increases mitochondrial Na⁺/Ca²⁺ NCLX exchanger expression, activity, function, or a combination thereof, thereby treating said disease.

52. The method of claim 51, wherein said agent is an isolated nucleic acid comprising a nucleic acid sequence encoding a mitochondrial Na⁺/Ca²⁺ NCLX exchanger, wherein said sequence shares at least 95% homology with SEQ NOs: 1-3 and wherein said contacting up- regulates expression, activity, function, or a combination thereof, of said exchanger.

53. The method of claim 51, wherein said cell is a preneoplastic cell, a neoplastic cell, an inflammatory cell, an infected cell, or any combination thereof.

54. The method of claim 51, wherein said cell is a T helper cell.