WO2016172952A1 - PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用 - Google Patents

PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用 Download PDF

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WO2016172952A1
WO2016172952A1 PCT/CN2015/078058 CN2015078058W WO2016172952A1 WO 2016172952 A1 WO2016172952 A1 WO 2016172952A1 CN 2015078058 W CN2015078058 W CN 2015078058W WO 2016172952 A1 WO2016172952 A1 WO 2016172952A1
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pi4kiiiα
protein
rbo
efr3a
inhibitor
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PCT/CN2015/078058
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English (en)
French (fr)
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黄福德
张潇
王文安
张乐
魏万国
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江苏挪贝肽医药科技有限公司
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Application filed by 江苏挪贝肽医药科技有限公司 filed Critical 江苏挪贝肽医药科技有限公司
Priority to PCT/CN2015/078058 priority Critical patent/WO2016172952A1/zh
Priority to KR1020197033850A priority patent/KR102344321B1/ko
Priority to EP16785990.9A priority patent/EP3290514A4/en
Priority to AU2016255474A priority patent/AU2016255474B2/en
Priority to PCT/CN2016/080907 priority patent/WO2016173562A1/zh
Priority to CN201680024899.8A priority patent/CN107849545B/zh
Priority to US15/570,681 priority patent/US11766420B2/en
Priority to RU2017141805A priority patent/RU2724493C2/ru
Priority to JP2018507766A priority patent/JP6755938B2/ja
Priority to KR1020177034632A priority patent/KR102048137B1/ko
Publication of WO2016172952A1 publication Critical patent/WO2016172952A1/zh
Priority to AU2020202847A priority patent/AU2020202847B2/en
Priority to JP2020142213A priority patent/JP2020203907A/ja
Priority to JP2022161004A priority patent/JP2023011586A/ja

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Definitions

  • the present invention was completed under the auspices of the Ministry of Science and Technology 973 project (2013CB530900) and the National Natural Science Foundation of China (81371400 and 81071026).
  • the present invention belongs to the field of medicine, and relates to a method for treating Alzheimer's disease by using a related inhibitor to down-regulate PI4KIII ⁇ kinase and a membrane protein complex formed thereof with RBO/EFR3/EFR3A/EFR3B protein and TTC7 protein;
  • the invention relates to a method for screening for drugs and therapeutic targets for the treatment of Alzheimer's disease depending on whether or not the A ⁇ secretion of the cells is promoted.
  • AD Alzheimer's disease
  • a ⁇ Alzheimer's disease
  • a ⁇ accumulates not only outside the cell but also in neurons.
  • a large amount of evidence suggests that A ⁇ accumulates in various intracellular organs of neurons and participates in pathological changes of AD such as synaptic disorder, amyloid plaque formation, and neuronal death.
  • oligo-A ⁇ which is thought to be the most destructive to synaptic and cognitive functions, is formed intracellularly and accumulates in the brain neurons of AD patients and APP transgenic mice or accumulates in the cell membrane. These membrane-coupled A ⁇ may be pinched on the cell membrane of the neuron or on the cell membrane of the cell. There may be many pathways leading to accumulation of A ⁇ in the accumulated neurons, such as extracellular A ⁇ endocytosis, decreased intracellular A ⁇ secretion and retention, autophagic vacuoles production and accumulation of A ⁇ , and decreased intracellular A ⁇ clearance.
  • a ⁇ accumulates inside and outside the cell
  • the concentration of A ⁇ 42 in the cerebrospinal fluid of patients with AD and those with early AD is reduced, about half that of the control population.
  • the concentration of A ⁇ 42 in cerebrospinal fluid and brain tissue fluid was also reduced in age, and A ⁇ dimer was not detected in brain tissue fluid.
  • the concentration of A [beta] 42 in CSF and brain tissue has been speculated to reduce fluid may be due to uptake of extracellular amyloid plaques, reducing A [beta] 42 secretion, induced by A [beta] 42 accumulation in the brain or neuronal cell membrane.
  • a ⁇ 42 can be inserted into the lipid membrane to bind to acidic phospholipids, and the combined acidic phospholipids promote A ⁇ 42 transition from the disordered configuration to the folded beta], resulting in the polymerization of A ⁇ 42 to lipid membranes or membrane; 2) culturing phosphatidylinositol 4,5 bisphosphate (phosphatidylinositol 4,5-phosphate in the cell membranes of cells, PIP2) level and the amount of cellular secretion of A ⁇ 42 was negatively correlated; 3) Drosophila neuronal dysfunction caused by A ⁇ 42 expression and learning and memory disorders can be repaired by the inhibition of PI3K.
  • the rolling blackout (rbo) gene in Drosophila encodes a membrane protein RBO with some homology to the diacylglycerol lyase.
  • RBO proteins play a role in phospholipid metabolism in Drosophila, light transmission in visual cells, synaptic transmission, and bulk endocytosis in bulk. RBO proteins are conserved from yeast to humans.
  • RBO homologous protein in yeast EFR3 and two homologous proteins of mouse (EFR3A and EFR3B) on the cell membrane with III ⁇ -type phosphatidylinositol 4 kinase (PI4KIII ⁇ ) and a scaffolding protein (called in mammals) Forming a complex for Tetratricopeptide repeat domain 7, TTC7, see Baird D, Stefan C, et al., 2008, J Cell Biol; Nakatsu F, Baskin JF, et al., 2012, J Cell Biol), thereby anchoring PI4KIII ⁇ On the cell membrane, and further regulating the levels of phosphatidylinositol 4-phosphate (PI 4 P) and PIP 2 on the cell membrane.
  • PI 4 P III ⁇ -type phosphatidylinositol 4 kinase
  • pancreatic neuronal expression of A ⁇ 42 fused to a secretion signal peptide results in accumulation of A ⁇ 42 and neurodegenerative changes in neurons.
  • the inventors expressed A ⁇ 42 containing a secretory signal peptide in a simple neural pathway of adult Drosophila, the giant fiber pathway (GF), and found that A ⁇ 42 accumulated in neurons and led to age-dependent (age-dependent) obstacles such as outstanding delivery failure and decreased athletic ability.
  • This Drosophila expressing A ⁇ 42 provides a good platform for studying the role of some genes that may be associated with AD in the accumulation of A ⁇ 42 in neurons and the synaptic dysfunction caused by it.
  • the inventors used this model to detect mutations or overexpression of genes rbo, PI4KIII ⁇ and ttc7, and the effects of commonly used PI4KIII ⁇ protein inhibitors on neurodegenerative diseases in this model.
  • the inventors also studied the atrophy of hippocampal neurons by a mouse homolog Efr3a, which knocks down the rbo gene, in APP/PS1 transgenic mice, and the use of the common PI4KIII ⁇ kinase small molecule inhibitor PAO to administer learning and memory.
  • the present invention discloses the use of genetic means or related inhibitors to inhibit down-regulation of PI4KIII ⁇ protein, RBO/EFR3/EFR3A/EFR3B membrane protein, TTC7 protein, and the amount of membrane protein complexes they form or their related enzyme activities can promote neuronal cells A ⁇ (especially A ⁇ 42 ) secretes and correspondingly reduces A ⁇ accumulation in neurons, thereby reducing neurological dysfunction in AD model Drosophila and mice.
  • the present invention thus discloses an important role of neuronal A ⁇ 42 secretion in the treatment of AD, and provides a novel strategy for the treatment of AD; at the same time, the present invention also provides a new drug for treating AD, and further screening for drugs for treating AD and The therapeutic target points to a new direction.
  • the invention relates to a novel method of treating Alzheimer's disease.
  • the present invention discloses a method for treating Alzheimer's disease using a PI4KIII ⁇ inhibitor, which may be an anti-PI4KIII ⁇ antibody, an inhibitory nucleotide specific for PI4KIII ⁇ , or A small molecule compound inhibitor specific for PI4KIII ⁇ .
  • the inhibitory nucleotide specific for PI4KIII ⁇ may be a nucleotide sequence as shown in SEQ ID NO: 6; the PI4KIII ⁇ inhibitor is selected from one or more of the following small molecule compound inhibitors; : PAO, a derivative of PAO, an analog of G1, A1, G1 or A1.
  • the invention also discloses a method of treating Alzheimer's disease using an RBO/EFR3/EFR3A/EFR3B inhibitor.
  • the RBO/EFR3/EFR3A/EFR3B inhibitor can be an anti-RBO/EFR3/EFR3A/EFR3B antibody.
  • the present invention also discloses a method for treating Alzheimer's disease using a PI 4 P inhibitor.
  • the PI 4 P inhibitor may be an anti-PI 4 P antibody, an OSH2-PH2X fusion protein, or an OSH2-2x-PH fusion protein.
  • the present invention is also a pharmaceutical composition for treating Alzheimer's disease comprising one or more of the following: a PI4KIII ⁇ inhibitor, a RBO/EFR3/EFR3A/EFR3B inhibitor, and a PI 4 P inhibitor, and optionally a pharmaceutical carrier.
  • the pharmaceutical composition may further comprise one or more anti-A ⁇ antibodies and/or a compound capable of scavenging extracellular A ⁇ polymers, wherein the compound capable of scavenging extracellular A ⁇ polymers is selected from the group consisting of: The saccharide HSH971 and its analogs, acamprosic acid and its analogs, and edaravone and its analogs.
  • the invention relates to a method of screening for a medicament for the treatment of Alzheimer's disease.
  • the invention discloses a PI4KIII ⁇ protein A method for screening for Alzheimer's disease drugs with a kinase activity as a therapeutic target, the method comprising the steps of: observing the effect of a candidate drug on the phosphokinase activity of the PI4KIII ⁇ protein, if the candidate drug is capable of inhibiting the phosphokinase activity of PI4KIII ⁇ , This drug candidate is a potential drug for the treatment of Alzheimer's disease.
  • the present invention also discloses a method for screening Alzheimer's disease drugs with the interaction of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein and PI4KIII ⁇ protein as therapeutic targets. Including the following steps: observing the effect of drug candidate on the interaction of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein and PI4KIII ⁇ protein, if the candidate drug can inhibit the mutual interaction of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein and PI4KIII ⁇ protein.
  • the role thereby reducing the formation of the RBO/EFR3/EFR3A/EFR3B-TTC7-PI4KIII ⁇ protein complex, indicates that the drug candidate is a potential drug for the treatment of Alzheimer's disease.
  • the present invention also discloses a method for screening an Alzheimer's disease drug with a level of PI 4 P on a cell membrane as a therapeutic target, the method comprising the steps of: observing Whether the drug candidate has an effect on the level of PI 4 P on the cell membrane, and if the drug candidate can inhibit the PI 4 P level on the cell membrane, it indicates that the drug candidate is a potential drug for treating Alzheimer's disease.
  • Figure 1 shows that mutation of the rbo gene alleviates neurological damage of Drosophila expressing A ⁇ arc , reduces the expression of PI4KIII ⁇ protein or attenuates the interaction of PI4KIII ⁇ protein with RBO protein.
  • Figure 2 shows down-regulation of PI4KIII ⁇ protein expression or drug inhibition of PI4KIII ⁇ enzyme activity to alleviate neurological impairment of Drosophila expressing A ⁇ arc .
  • Figure 3 shows that down-regulation of RBO/PI4KIII ⁇ protein expression or function reduces the accumulation of A ⁇ in neurons.
  • Figure 4 shows that down-regulation of RBO/PI4KIII ⁇ protein expression or function promotes secretion of A ⁇ 42 .
  • Figure 5 shows knockdown of Efr3a gene in hippocampal CA3 and in APP/PS1 and control mice.
  • Figure 6 shows the effect of PAO treatment on learning and memory ability of APP/PS1 mice and control mice, as well as CSF and brain cell membrane-coupled A ⁇ 42 levels.
  • Figure 7 shows expression rbo mutation alleviate neurological injury Drosophila A ⁇ 42.
  • Figure 8 shows the effect of rbo gene and shibire gene mutations on the motility and longevity of Drosophila overexpressing A ⁇ arc or Drosophila Tau protein, respectively.
  • Figure 9 shows the effect of mutations in the rbo S358A gene and the itpr SV35 gene on expression of A ⁇ arc fruit flies.
  • Figure 10 shows the effect of the outer cells 42 and A [beta] rbo PI4KA genes and gene mutation gene knockdown Efr3a N2a cells on the endocytosis of A ⁇ arc Drosophila transcription expression of A ⁇ arc.
  • Figure 11 shows the efficiency of knockdown of the Efr3a gene or PI4KA gene in HEK293 cells or mouse primary cultured hippocampal neurons.
  • Figure 12 shows that insertion of a transposon into one copy of the PI4KA gene of APP/PS1 mice significantly improved learning and memory in mice.
  • Figure 13 shows that PI 4 P promotes the formation of oligomers of A ⁇ 42 in liposomes.
  • Figure 14 shows the effect of PAO on APP expression and ⁇ , ⁇ , ⁇ secretase activity.
  • Figure 15 shows the effect of ttc7 gene mutation and overexpression on neurological impairment of A ⁇ arc Drosophila.
  • rbo/Efr3/Efr3a/Efr3b gene refers to a rbo gene derived from Drosophila, a yeast-derived Efr3, or a mammalian Efr3a gene and an Efr3b gene; the term “RBO/EFR3” as used in the present invention.
  • the /EFR3A/EFR3B protein refers to a protein derived from a fruit fly-derived rbo gene, a yeast-derived Efr3 gene, or a mammal-derived Efr3a/Efr3b gene.
  • PI4KIII ⁇ /PI4KA gene refers to a fruit fly or breastfeeding.
  • the term “PI4KIII ⁇ protein” as used in the present invention refers to a protein encoded by the PI4KIII ⁇ /PI4KA gene in a fruit fly or a mammal.
  • ttc7 gene refers to a ttc7 gene derived from a mammal
  • TTC7 protein refers to a protein encoded by the ttc7 gene in the aforementioned mammal.
  • inhibitor refers to a substance capable of reducing, reducing, or eliminating the amount of a target, a specific function, and a specific property.
  • the target may be a protein, a polypeptide, a nucleic acid, or the like, and the inhibitor may directly or indirectly act on the number, specific function, and specific property of the target, and the number and specific function of the target may be made. And the corresponding properties are correspondingly reduced, reduced or eliminated.
  • the inhibitor may be a protein, a polypeptide, a nucleic acid, a small molecule compound or the like.
  • PI4KIII ⁇ inhibitor means the stability of the PI4KIII ⁇ protein capable of reducing, reducing, and eliminating the expression, transcription, translation, and/or formation thereof of the PI4KIII ⁇ /PI4KA gene, and the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B and the binding ability of TTC7 protein, and various factors such as phosphokinase activity, including but not limited to inhibitory nucleotides specific for PI4KIII ⁇ /PI4KA gene, anti-PI4KIII ⁇ protein antibody, and ability to inhibit PI4KIII ⁇ kinase An active small molecule compound, and/or various substances capable of inhibiting the interaction of the PI4KIII ⁇ protein with other membrane proteins.
  • RBO/EFR3/EFR3A/EFR3B inhibitor refers to an RBO/ capable of inhibiting, reducing, or eliminating the expression, transcription, translation, and/or formation of the rbo/Efr3/Efr3a/Efr3b gene.
  • EFR3/EFR3A/EFR3B protein The stability of EFR3/EFR3A/EFR3B protein, its ability to bind to PI4KIII ⁇ protein, and other substances, including but not limited to inhibitory nucleotides specific for rbo/Efr3/Efr3a/Efr3b gene, anti-RBO/EFR3
  • the antibody of the /EFR3A/EFR3B protein and various substances capable of inhibiting the formation of a complex of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein The antibody of the /EFR3A/EFR3B protein and various substances capable of inhibiting the formation of a complex of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein.
  • TTC7 inhibitor refers to a TTC7 protein which is capable of reducing, reducing, and eliminating the expression, transcription, translation, and/or formation of the ttc7 gene, and the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B.
  • Various substances in combination including but not limited to inhibitory nucleotides specific for the ttc7 gene, anti-TTC7 protein antibodies, and/or inhibiting the interaction of the TTC7 protein with the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B Various substances, etc.
  • PI 4 P inhibitor refers to a substance capable of inhibiting, reducing, and eliminating the level of PI 4 P on a cell membrane, including but not limited to an anti-PI 4 P antibody, and capable of being specific to PI 4 P. Binding OSH2-PH2X fusion protein or OSH2-2x-PH fusion protein, and the like.
  • antibody refers to any immunoglobulin or intact molecule that binds to a particular epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of intact antibodies, as long as these fragments or portions retain the antigen binding ability of the parent antibody.
  • anti-PI4KIII ⁇ antibody refers to a monoclonal antibody, a polyclonal antibody, a single-chain antibody, and an immunologically active fragment or portion thereof capable of specifically binding to a PI4KIII ⁇ protein, or a functional variant or a functional fragment thereof.
  • terms such as “PI4KIII ⁇ antibody”, “anti-PI4KIII ⁇ antibody”, and “antibody against PI4KIII ⁇ ” are used interchangeably.
  • “functional variant” refers to a protein or polypeptide of the invention having one or more amino acids altered in its amino acid sequence.
  • the alteration may be a "conservative” change (wherein the substituted amino acid has similar structural or chemical properties) or a “non-conservative” change; similar changes may also include amino acid deletions or insertions or both.
  • alterations in these amino acid residues or deletions or insertions of amino acids should not substantially alter or disrupt the biological or immunological activity and function of the original amino acid sequence.
  • “functional fragment” refers to any portion of a protein or polypeptide of the invention that retains substantially similar or identical protein or polypeptide (ie, "parent” protein or polypeptide) as part of it. Biological or immunological activity and function.
  • inhibitory nucleotide refers to a nucleotide compound capable of binding to and inhibiting expression of a specific gene.
  • Typical inhibitory nucleotides include, but are not limited to, antisense oligonucleotides, triple helix DNA, aptamers, ribozymes, short-suppressed ribonucleosides Glycosylate (siRNA), short hairpin RNA (shRNA) and microRNA. These nucleotide compounds are capable of binding to the specific gene with greater affinity than other nucleotide sequences, thereby inhibiting the expression of a particular gene.
  • small molecule compound refers to an organic compound having a molecular weight of less than 3 kilodaltons, which may be natural or chemically synthesized.
  • derivative refers to a compound produced by modifying a parent organic compound by one or more chemical reactions, which has a similar structure to the parent organic compound and has a similar effect in function.
  • analog refers to an organic compound which is not necessarily obtained by chemical modification of a parent organic compound, but which is structurally similar to a parent organic compound and functionally It also has a similar effect.
  • AD Alzheimer's disease
  • senile neurodegenerative disease in which progressive learning and memory disorders are prominent clinical symptoms.
  • Most AD patients have extracellular beta-amyloid plaques in the middle and advanced stages, and there are neurofibrillary tangles composed of Tau protein or synaptic and nerve cell loss.
  • the disease can exist in both humans and animals, such as dogs.
  • a ⁇ refers to a series of polypeptides which are between 38-48 amino acids in length and which are cleaved by secretases from Amyloid Precursor Protein (APP), mainly A ⁇ 38 .
  • a ⁇ can also be cleaved by other protein cleavage enzymes expressed by a method of infecting cells with a transgene or a viral vector, for example, the N-terminus of A ⁇ can be derived from Drosophila.
  • Secretion signal peptide of the protein encoded by the necrotic gene (amino acid sequence: MASKVSILLLLTVHLLAAQTFAQDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (SEQ ID NO: 1) or secretion signal peptide derived from rat Proenkephalin precursor (pre-proenkephalin) (amino acid sequence: MAQFLRLCIWLLALGSCLLATVQA (SEQ ID NO: 2) ))
  • a ⁇ secretion refers to a process in which A ⁇ is discharged extracellularly via a cell membrane after intracellular or cell membrane production, which may result in a decrease in intracellular A ⁇ accumulation.
  • the secretion of A ⁇ 42 specifically refers to A ⁇ 42 in the cell or after discharged to the cell membrane via extracellular membrane process, the process may lead to reduced intracellular accumulation of A ⁇ 42.
  • the term "therapeutic target” as used in the present invention refers to various substances which can be used for the treatment of a certain disease and their action targets in an animal or a human body.
  • the action of the substance on the target of the action can have an effect of treating the disease.
  • the substance may be a protein, a polypeptide, a nucleic acid, a small molecule compound or the like, and the target may be a certain gene (including a specific sequence of the gene), a certain protein (including a specific site of the protein),
  • a substance entity such as a certain protein complex (including a specific binding site thereof) may also be a certain characteristic of the aforementioned gene and/or protein, a certain function, a relationship with a surrounding substance, an environment, etc., as long as The substance can affect the gene, protein, protein complex, or its specific characteristics, functions, relationships, etc., thereby functioning to treat the disease.
  • treating refers to reversing, alleviating or inhibiting the progression of a disease to which the term is applied, or one or more symptoms of a disease.
  • the term also includes preventing the disease, including preventing the onset of the disease or any symptoms associated therewith, as well as reducing the severity of the condition or any condition prior to its onset.
  • the terms “inhibiting,” “weakening,” “down-regulating,” “eliminating,” and the like, are meant to mean a reduction or reduction in quantity or degree. This reduction or reduction is not limited to any amplitude as long as this trend is exhibited. For example, this kind reduces or reduces the amount relative to the original Or the degree can be 100%, it can be 50%, or it can be 1% or less.
  • the present invention discloses down-regulating the expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein or PI4KIII ⁇ protein, weakening the interaction between RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII ⁇ protein, and inhibiting the enzyme activity of PI4KIII ⁇ .
  • a series of behaviors can alleviate defects such as synaptic dysfunction and loss of nerve cells expressing A ⁇ 42 with increasing age, and disclose that these effects are promoted by promoting the secretion of A ⁇ (especially A ⁇ 42 ) in nerve cells. Reduce the accumulation of A ⁇ (especially A ⁇ 42 ) in nerve cell membranes or nerve cells.
  • the inventors found that knockdown of the Efr3a gene can reduce dendritic and spine atrophy of hippocampal neurons in APP/PS1 double transgenic mice, and a commonly used PI4KIII ⁇ protein inhibitor, phenylarsenic oxide (Phenylarsine Oxide). , PAO) can significantly improve the learning and memory ability of APP/PS1 mice, and reduce the content of A ⁇ 42 , especially polymerized A ⁇ 42 , which is coupled to the cell membrane of brain tissue, although the process is accompanied by A ⁇ 42 in cerebrospinal fluid. The content is increased.
  • PAO phenylarsenic oxide
  • the inventors have found that genetically downregulating the expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII ⁇ protein in cells or neurons, or preventing them from forming protein complexes, reduces the membrane distribution of PI4KIII ⁇ protein, or inhibits
  • the phosphokinase activity of PI4KIII ⁇ protein can promote the secretion of A ⁇ 42 in cells or neurons, reduce the accumulation of A ⁇ 42 in neurons, alleviate AD-related neurodegenerative changes and dysfunction, but at the same time, whether it is A ⁇ 42
  • the expression level of APP or the activity of ⁇ , ⁇ and ⁇ secretase of APP was not significantly affected.
  • PI 4 P a product of the PI4KIII ⁇ protein, promotes polymerization of A ⁇ 42 monomers in liposomes to form multimers, and the promotion is better than the precursor of PI 4 P (PI) and its derivative PI 4 , The promotion of 5 P is much stronger.
  • a ⁇ (including A ⁇ 42 ) is produced on the plasma membrane or in intracellular organs, and the resulting A ⁇ may be released by passive release, exocytosis, lysosomal-mediated release, or other undiscovered pathways.
  • Secreted cells The inventors believe that A ⁇ (including A ⁇ 42 ) is produced on the plasma membrane or in intracellular organs, and the resulting A ⁇ may be released by passive release, exocytosis, lysosomal-mediated release, or other undiscovered pathways. Secreted cells. However, no matter where A ⁇ is produced, how it is secreted, the cytoplasmic membrane should be the last pathway that A ⁇ must pass after leaving the cell. Due to the hydrophobicity of A ⁇ , on the cytoplasmic membrane, A ⁇ is inserted into the hydrophobic fatty acid chain on the one hand, and on the other hand with phosphatidylinositol (especially PI 4 P in phosphorylated phosphatidylinositol) and other acidic phospholipids
  • PI 4 P is one of the main components of phosphorylated phosphatidylinositol on the cell membrane, and its promoting effect on the formation of A ⁇ polymer is stronger than that of PI and PI 4,5 P.
  • the inventors have found that the promotion effect of PI 4 P on the formation of a polymer of A ⁇ 42 in a lipid is in a dose-dependent manner. Down-regulation of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein or PI4KIII ⁇ protein expression, inhibition of their formation of a complex, or inhibition of the kinase function of the PI4KIII ⁇ protein will reduce the amount of PI 4 P produced on the cell membrane.
  • a ⁇ 42 in Drosophila cells activates PI3K and its associated PI3K/Akt signaling pathway, thereby inducing AD-related synaptic impairment and long-term memory loss; accordingly, related studies suggest inhibition of PI3K The activity can also be used as a means of treating AD.
  • the inventors believe that the accumulation of A ⁇ 42 in cells is not caused by the activation of the PI3K/Akt signaling pathway associated with phosphatidylinositol kinase (including PI3K), but more directly due to the plasma membrane.
  • the phosphatidylinositol kinase involved in the present invention is mainly PI4K, and is not PI3K involved in the PI3K/Akt signaling pathway.
  • PI4K secreted A ⁇ 42 having high specificity but not sensitive PI3K phosphatidylinositol kinase inhibitors, for example PAO, i.e. at lower concentrations may be effective in promoting cell.
  • the present invention discloses that in order to achieve the purpose of treating AD, a method of promoting A ⁇ secretion, particularly A ⁇ 42 secretion, may be employed to reduce accumulation of A ⁇ (including A ⁇ 42 ) in or on nerve cells.
  • a ⁇ including A ⁇ 42
  • the increased secretion of A ⁇ cannot be attributed to an increase in the expression level of APP or an increase in the production of A ⁇ caused by the activities of ⁇ , ⁇ and ⁇ secretase.
  • promoting A ⁇ secretion by nerve cells can take many forms, including impairing the binding or interaction of A ⁇ with sugar, lipids, and proteins on the cell membrane.
  • the present invention also discloses that the regulation of RBO/EFR3/EFR3A/EFR3B protein, the amount of TTC7 protein and PI4KIII ⁇ protein and their ability to form complexes, and the regulation of the phosphokinase activity of PI4KIII ⁇ protein can reduce the formation of A ⁇ on the cell membrane.
  • the polymer which promotes the secretion of A ⁇ (especially A ⁇ 42 ) in cells, so these proteins and the interrelationship of these proteins can constitute potential therapeutic targets for the treatment of AD.
  • the RBO/EFR3/EFR3A/EFR3B inhibitors include, but are not limited to, inhibitory nucleotides of the rbo/Efr3/Efr3a/Efr3b gene (including antisense RNA, siRNA or miRNA, etc.), and anti-RBO/EFR3/EFR3A/EFR3B Protein antibodies, etc.
  • inhibitory nucleotides of the rbo/Efr3/Efr3a/Efr3b gene and methods for their preparation have been disclosed (for example, see www.genecards.org, and existing products: ORIGENE, Cat. #SR308056 and Cat.#TR303768).
  • methods for preparing anti-RBO/EFR3/EFR3A/EFR3B antibodies have also been disclosed (for example, see www.genecards.org, and existing products: Novus, Cat. #NBP1-81539; Thermo Fisher Scientific Cat.# PA5-24904).
  • the expression, transcription, and translation of the PI4KIII ⁇ /PI4KA gene are regulated, and the stability of the PI4KIII ⁇ protein encoded by the PI4KIII ⁇ /PI4KA gene is regulated, and the protein and membrane proteins RBO/EFR3/EFR3A/EFR3B and TTC7 are regulated.
  • the ability of a protein to form a protein complex, as well as the enzyme activity that regulates the phosphokinase, can also be used as a means of treatment for AD.
  • the anti-PI4KIII ⁇ protein antibody can inhibit the formation of a membrane protein complex between the PI4KIII ⁇ protein and the membrane protein, and a small molecule compound inhibitor capable of inhibiting the kinase activity, etc., can be used for the treatment of AD. .
  • the inhibitor is a small molecule compound, for example, PAO (phenylarsenic oxide or Phenilarsine Oxide), a derivative of PAO, A1, G1, or A1 and an analog of G1. More preferably, the inhibitor may be PAO or a derivative thereof.
  • PAO phenylarsenic oxide or Phenilarsine Oxide
  • the inhibitor may be PAO or a derivative thereof.
  • PAO is a small molecule compound having an arsenic oxide group and a benzene ring as a basic structure, and has strong inhibition of the phosphokinase activity of the PI4KIII ⁇ protein. effect.
  • the chemical structure of PAO is:
  • Phenyl arsenic oxide (oxo(phenyl)arsane)
  • A1 and G1 are small molecule compound inhibitors of the PI4KIII ⁇ protein, and have a similar structure.
  • the chemical structure of A1 is:
  • A1 and G1 Methods for the preparation of A1 and G1 are disclosed (for example, see Bojjireddy, N., et al. (2014), JBC 289: 6120-6132 and Leivers, AL, et al. (2014), JMC 57: 2091-2106).
  • structural analogs of A1 and G1 can also be used for the treatment of AD as long as they have a function of inhibiting the phosphokinase activity of the PI4KIII ⁇ protein.
  • methods for preparing such structural analogs are also disclosed.
  • TTC7 inhibitor includes, but is not limited to, an inhibitory nucleotide of the ttc7 gene (including antisense RNA, siRNA or miRNA, etc.), an antibody against the TTC7 protein, and the like.
  • downregulating the expression of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein, or preventing them from forming a complex to reduce the membrane distribution of the PI4KIII ⁇ protein, or inhibiting the phosphokinase activity of the PI4KIII ⁇ protein substantially results in The decrease in PI 4 P on the cell membrane promotes A ⁇ secretion from the cells. Accordingly, those of ordinary skill in the art understand, any time or inhibitor reduce the number of method PI 4 P level on cell membrane, in turn, can reduce the role of A ⁇ multimers formed in the cell membrane, it can also play the treatment of AD.
  • the PI 4 P inhibitors may be capable of binding to PI 4 P specific antibodies or other molecules.
  • Current methods for the preparation of anti-PI 4 P antibodies have been disclosed (see Brown BK and Karasavass N, et al., 2007, J virol; Wassef NM and Roerdink F, et al. 1984, Mol Immuol).
  • it can be a broad-spectrum neutralizing antibody 4E10 and other anti-PI4P antibodies of human origin.
  • the above various substances provided by the present invention may be used for the treatment of AD or a substance having therapeutic AD (collectively referred to as "the substance of the present invention"), including but not limited to an anti-RBO/EFR3/EFR3A/EFR3B antibody, an anti-PI4KIII ⁇ antibody, and an anti-PI4KIII ⁇ antibody.
  • TTC7 antibody anti-PI 4 P antibody
  • inhibitory nucleotide specific for rbo/Efr3/Efr3a/Efr3b gene inhibitory nucleotide specific for PI4KIII ⁇ /PI4KA gene
  • inhibitory effect on phosphokinase of PI4KIII ⁇ protein The small molecule compounds and the like may all be isolated, purified, synthesized, and/or recombinant.
  • compositions such as pharmaceutical compositions.
  • the invention provides pharmaceutical compositions comprising any of the foregoing antibodies, inhibitory nucleotides, or/and small molecule compounds, and the corresponding pharmaceutically acceptable carriers.
  • the pharmaceutical composition comprising any of the substances of the invention may comprise more than one substance of the invention, for example: antibodies and small molecule compounds, inhibitory nucleotides and antibodies, or two or more different antibodies or small molecule compounds and the like.
  • the pharmaceutical composition may also include a combination with another or more pharmaceutically active agents or drugs.
  • an anti-A ⁇ antibody drug such as Bapineuzumab, or a compound that binds to A ⁇ or ⁇ amyloid plaques outside the brain's nerve cells, blocks A ⁇ polymerization or promotes polymerization of A ⁇ , such as marine sulfuric acid, may be included.
  • Oligosaccharides HSH971 and its analogs, tramiprosate and its analogs, Edaravone and its analogs, and the like may be included.
  • the pharmaceutical composition promotes the secretion of A ⁇ from the neurons on the one hand, and promotes the clearance of A ⁇ outside the neurons on the one hand, thereby achieving a better therapeutic effect on AD.
  • the invention also discloses a novel method for screening for a drug or therapeutic target for the treatment of AD.
  • the design of this method is based on the novel discovery of the present invention described above, that is, the manner of promoting secretion of A? 42 can reduce the accumulation of intracellular A? 42 , thereby alleviating and preventing AD-related neurodegeneration and dysfunction. Therefore, the criteria for screening candidate drugs or therapeutic targets is to achieve the effect of promoting A ⁇ secretion of cells after the drug is administered or modulating the therapeutic target, especially the secretion of A ⁇ 42 , and the increased A ⁇ secretion cannot be attributed to the up-regulation.
  • the expression level of APP, or the change in the activity of ⁇ , ⁇ or ⁇ secretase leads to an increase in the production of A ⁇ .
  • the regulation of the therapeutic target refers to direct or indirect action of the therapeutic target with the related substance, thereby causing a change in the function, properties or correlation with the surrounding environment of the therapeutic target, thereby enabling Produces or triggers the corresponding effect of promoting A ⁇ secretion from cells, especially the secretion of A ⁇ 42 .
  • the cell line that can be used for the screening test can be a eukaryotic cell line of mammals, insects, etc., such as: HEK293, COS7, N2a, SH. -SY5Y, S2 and sf9, etc.
  • the method comprises detecting whether the candidate drug can reduce A ⁇ accumulation in the cell membrane or cells, especially the accumulation of A ⁇ 42 , thereby selecting an effective drug for treating Alzheimer's disease.
  • the detection assay for whether the A ⁇ 42 secretion is increased can be carried out using a cell line overexpressing APP (such as HEK293, COS7, N2a, SH-SY5Y cell lines stably transfected with human APP), or using a fruit fly model. Preferably, it is a third instar larva tissue of Drosophila. Whether the A ⁇ 42 secretion is increased or not can be detected by an immunoassay method, including an enzyme-linked immunosorbent assay (ELISA) or an electrochemiluminescence immunosorbent assay (ECLIA) method.
  • ELISA enzyme-linked immunosorbent assay
  • ELIA electrochemiluminescence immunosorbent assay
  • the method for screening for a drug for treating AD or for modulating a target may comprise first observing the effect of the candidate drug or the application of the regulatory target on the activity of the PI4KIII ⁇ enzyme, if the candidate drug or the application of the regulatory target enables the detection system
  • a negative change in the effect of PI4KIII ⁇ kinase, ie, attenuation of PI4KIII ⁇ enzyme activity or a decrease in PI4P levels on the cell membrane indicates that the drug, agent or target is a potential drug or regulatory target for the treatment of AD.
  • it is further tested whether it causes a decrease in the accumulation of intracellular A ⁇ (especially A ⁇ 42 ) and whether it promotes the secretion of A ⁇ into the extracellular. With such a method, the screening efficiency of candidate objects can be greatly improved.
  • the screening for a medicament for the treatment of AD or The method of treating the target can reduce the amount of PI4KIII ⁇ protein on the cell membrane by directly detecting whether the application of the candidate drug or the regulation of the therapeutic target can cause the PI4KIII ⁇ protein originally located in the cell membrane to be distributed to the cytoplasm, thereby reducing the A ⁇ on the cell membrane. Judging by the conversion of monomers to polymers and increased secretion to the extracellular.
  • the fluorescently labeled PI4KIII ⁇ such as fluorescent protein-labeled PI4KIII ⁇ (GFP-PI4KIII ⁇ )
  • GFP-PI4KIII ⁇ fluorescent protein-labeled PI4KIII ⁇
  • the fluorescently labeled PI4KIII ⁇ can be selectively observed on the cell membrane to observe whether the fluorescently labeled PI4KIII ⁇ is transferred from the cell membrane to the cytosol.
  • the method of screening for a drug or therapeutic target for the treatment of AD can also be carried out by a method comprising detecting the interaction of a protein with a protein, such as co-immunoprecipitation.
  • a protein such as co-immunoprecipitation.
  • the application of a drug candidate or the regulation of a therapeutic target can attenuate the interaction of the RBO/EFR3/EFR3A/EFR3B protein with the TTC7 protein and the PI4KIII ⁇ protein, ie, the ability of the drug or target to impair the ability of the PI4KIII ⁇ protein to form a membrane egg complex white.
  • the transformation of the A ⁇ monomer on the cell membrane to the polymer is reduced, and the secretion to the extracellular is increased.
  • the method for screening a drug or a therapeutic target for treating AD can also be performed by directly detecting the application of the drug candidate or whether the regulation of the therapeutic target can lower the PI 4 P level of the cell membrane.
  • fluorescence-microscopic, confocal or two-photon microscopy can be used to observe fluorescently labeled molecules that specifically bind to PI 4 P on the cell membrane, such as fluorescent protein-labeled OSH2-PH2X or OSH2-2x-PH fusion protein, on the cell membrane. Whether it is reduced, or whether it is transferred from the cell membrane to the cytosol.
  • Example 1 Drosophila strain and genetic method
  • the standard medium was circulated alternately under the conditions of 12 hours of light and 12 hours of darkness, and cultured at a constant temperature of 25 °C.
  • the present invention employs the following transgenic Drosophila strains: rbo S358A , [UAS] A ⁇ arc , [UAS] A ⁇ 42 , [UAS] dtau, [UAS] mCD8-gfp (provided by Dr. Z. Wang), [UAS] shibire tsl (by Dr.A Guo), [Gal4] A307 ( supplied by Dr.O'Kane).
  • the rbo S358A gene is a transgene constructed by site-directed mutagenesis of wild-type genomic DNA containing the rbo gene, and its expression is driven by the pre-driver of the rbo gene itself.
  • [Gal4]A307 expresses the transcription factor Gal4, which is used to drive the [UAS]A ⁇ arc , [UAS]A ⁇ 42 , [UAS]dtau, [UAS]mCD8-gfp, [UAS]shibire ts1 transgene in the giant fiber pathway of Drosophila ( Giant Fiber system) Dynamin expressing neurons of A ⁇ arc , A ⁇ 42 , dTau, mCD8-GFP or temperature-sensitive mutations in neurons and a small number of other neurons.
  • the Drosophila mutants used were: rbo ts1 (temperature-sensitive missense mutation), rbo 2 (knockout mutation), itpr sv35 (nonsense mutation, Bloomington Stock #30740), PI4KIII ⁇ def (deletion of PI4KIII ⁇ gene and its vicinity) DNA mutations, Bloomington Stock #9518), PI4KIII ⁇ GS27 and PI4KIII ⁇ GJ86 (both nonsense mutations).
  • all transgenic and mutant fruit flies were backcrossed with a wild isogenic line (isogenic w 1118 , Bloomington stock #5905) for 5 generations before use. the above.
  • Drosophila (A ⁇ 42 or A ⁇ arc Drosophila) expressing wild-type or arctic mutant A ⁇ 42 in the GF pathway exhibits accumulation of A ⁇ 42 in neurons and age-dependent synaptic transmission. Failure and shortened life. These fruit flies also show a decline in age-dependent crawling ability.
  • two mutations of the rbo gene, the missense mutation (rbo ts1 ) and the knockout mutation (rbo 2 ) were introduced into the A ⁇ arc flies. They were tested for their effects on synaptic transmission, crawling ability, and longevity.
  • Each group of fruit flies contains 1-2 lines, of which "ctrl” refers to the wild type control fruit fly with the [Gal4]A307 transgene; “rbo ts1/+ “ and “rbo 2/+ “ means One copy of [Gal4]A307 transgenic rbo ts1/+ and rbo 2/+ heterozygous Drosophila; "A ⁇ arc “ refers to a fruit fly with ([Gal4]A307/[UAS]A ⁇ arc double transgene; "A ⁇ arc -rbo ts1/+ ” and “A ⁇ arc -rbo 2/+ ” are respectively referred to as [Gal4]A307-rbo ts1 /[UAS]A ⁇ arc and [Gal4]A307-rbo 2 /[UAS]A ⁇ arc . Drosophila does not express A ⁇ arc and is classified as “non-A ⁇ flies”,
  • Example 2 Detection of rbo gene mutation by synaptic transmission specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
  • the excitatory junction potential (EJP) method of intracellular excitability was recorded in the escape pathway.
  • Adult female flies of a specific age were fixed on a glass slide with a low melting wax, tackiwax (Boekel Scientific), with the abdomen down.
  • the recording system includes a reference electrode on the abdomen, two stimulating electrodes inserted into the eye, and a recording electrode inserted into the longitudinal muscle cells of the back. Electrical stimulation (100 Hz, 50 pulses) was given to both eyes.
  • the stimulation intensity is 5-20 Volts with a duration of 0.2 ms, which is about 150% of the threshold stimulation intensity.
  • Figure 1 a representative record of brain stimulation-induced EJP in four groups of Drosophila at different ages (see Figure 1(a)), and a quantitative analysis of the success rate of induced EJPs (see Figure 1(b)) .
  • the rbo mutation significantly inhibits age-dependent neurosynaptic transmission failure caused by A ⁇ arc .
  • high-frequency electrical stimulation 100 Hz, 50 pulses
  • EJP endplate potential
  • Example 3 Detection of rbo gene mutation by creep test specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
  • the ability to climb the tube was measured by measuring the height of 10 fruit flies crawling up from the bottom of an upright transparent plastic tube for 7 seconds.
  • a highly reproducible fruit fly creep test capability test device was developed.
  • the device comprises: 1) a rectangular metal frame (32 cm wide and 21 cm high), 10 transparent plastic tubes (inner diameter 2.1 cm, height 19.0 cm) vertically fixed in the frame; 2) an electric motor for driving the metal frame Moving in the vertical direction; 3) a stepper drive to control the working rhythm of the electric motor to make the metal frame every 1 minute Continuously move up and down four times each time, each time moving up and down a predetermined height; 4) a video recorder for recording the creeping process; 5) a set of analysis software for analyzing the crawling position of the fruit fly at a specific moment in the video.
  • Example 4 Analysis of rbo gene mutations by lifespan specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
  • Each genotype of 100 or 200 fruit flies was equally distributed to 5 or 10 tubes containing standard Drosophila food and dry yeast, and cultured at 25 °C. Drosophila was exchanged into tubes containing new food and dry yeast every 3 days and the number of dead fruit flies was recorded. Survival was finally calculated using SPSS 11 Kaplan-Meier software.
  • the rbo gene mutation has an effect on dyskinesia (left) and premature death caused by overexpression of Drosophila Tau protein in the Drosophila giant fiber pathway.
  • the shibire gene mutation has an effect on dyskinesia (left) and premature death caused by overexpression of A ⁇ arc in the Drosophila giant fiber pathway.
  • the toxic effects of rbo gene mutations against A ⁇ 42 cannot be attributed to a general effect on the accumulation of toxic proteins in neurons, as mutations in the rbo gene did not improve the lifespan of Drosophila overexpressing Tau protein (Fig. 8a).
  • the toxic effect of the rbo gene mutation against A ⁇ 42 cannot be attributed to a general effect that may be based on synaptic function or down-regulation of endocytosis, since the introduction of a shibire gene mutation (Shibire ts1 ) into A ⁇ arc Drosophila does not alleviate A ⁇ arc
  • the phenomenon of premature death of fruit flies Fig. 8b.
  • mutations in the shibire ts1 gene also cause temperature-dependent synaptic transmission, bulk endocytosis, and motor function changes.
  • Examples 1-4 the results show that mutation or deficiency of the rbo gene can specifically improve neurodegenerative diseases expressing wild-type and mutant A ⁇ 42 Drosophila.
  • Example 5 Detection of PI4KIII ⁇ enzyme lacking or inhibiting interaction with RBO protein by immunoprecipitation to alleviate nerve damage in A ⁇ arc Drosophila
  • Tris buffer formulation was as follows: 50 mM Tris, 50 mM KCl, 1 mM EDTA, 1% cocktail protease inhibitor (Calbiochem), adjusted to pH 7.4.
  • the tissue homogenate was centrifuged at 10000 g for 10 minutes, and the supernatant was taken, and about 1 ⁇ g of a mouse-derived Drosophila RBO monoclonal antibody or a rabbit polyclonal antibody against Drosophila PI4KIII ⁇ was used for co-immunoprecipitation and immunoblotting.
  • the above two antibodies were constructed in cooperation with Shanghai Abmart and China Abgent.
  • RBO antibody polypeptide construct used is 251 th -500 th amino acid subtype C RBO Drosophila protein, polypeptide construct is against the Drosophila PI4KIII ⁇ NH2-KRSNRSKRLQYQKDSYC-CONH2 (SEQ ID NO: 3).
  • the dilution ratio of the anti-Drosophila RBO and PI4KIII ⁇ antibodies was 1:2000.
  • Anti-Drosophila RBO and PI4KIII ⁇ antibodies were verified by head tissue homogenate of wild-type and corresponding homozygous mutants, respectively.
  • RBO is a putative diacylglycerol (DAG) lyase
  • DAG diacylglycerol
  • the homologous protein of the RBO protein in yeast cells and mice binds to the PI4KIII ⁇ protein and forms a complex on the cell membrane. Consistent with this, the RBO protein specifically immunoprecipitated with the Drosophila PI4KIII ⁇ protein (Fig. 1e).
  • removal of one copy of the rbo gene (rbo 2/+ ) in A ⁇ arc -rbo Drosophila significantly reduced the expression levels of RBO and PI4KIII ⁇ proteins (Fig. 1f), while the rbo ts1/+ gene mutation did not decrease.
  • the expression levels of RBO protein and PI4KIII ⁇ protein but significantly impaired the interaction between RBO protein and PI4KIII ⁇ protein (Fig. 1g). It is worth noting that the two rbo gene mutations did not alter the transcription of the PI4KIII ⁇ gene (Fig. 1h).
  • a ⁇ arc Drosophila deletions are introduced into chromosomes (chromosome DNA fragment containing the deleted gene PI4KIII ⁇ , Pi4k def/+ ) and a nonsense mutation of PI4KIII ⁇ (PI4KIII ⁇ GS27/+ ).
  • the A ⁇ staining method in the Drosophila central system is as follows.
  • the antigenic determinants were exposed by washing with PBS for 30 minutes, followed by treatment with formic acid (70% in water) for 45 minutes, and then washed repeatedly with PBS-0.25% Triton-5% BSA.
  • the primary antibody (6E10, 1:100 dilution) was incubated for 10-12 hours at 4 °C.
  • the nerve damage caused by the expression of A ⁇ arc in the GF pathway was attributed to the accumulation of intracellular A ⁇ amyloid.
  • the introduction of the uas-mCD8-gfp transgene into A ⁇ arc Drosophila further confirmed the accumulation of A ⁇ amyloid in neurons.
  • the uas-mCD8-gfp transgene expresses the mCD8-GFP fluorescent protein localized to the cell membrane, which is driven by the same driver as A ⁇ arc , so neurons expressing A ⁇ arc are labeled by GFP, confocal microscopy It was revealed that most of the A ⁇ immunostaining signals co-localized with the GFP signal (Fig. 3(a)), thus confirming the phenomenon of A ⁇ accumulation in neurons in this Drosophila model.
  • a ⁇ immunostaining was performed in A ⁇ arc , A ⁇ arc -rbo, and A ⁇ arc -PI4KIII ⁇ Drosophila.
  • the A ⁇ immune response signal of A ⁇ arc -rbo and A ⁇ arc -PI4KIII ⁇ Drosophila was found to be significantly weaker than that of A ⁇ arc Drosophila (Fig. 3(b)).
  • the A ⁇ 42 Human ELISA Kit (invitrogen) was used for the ELISA and experiments were performed according to the instructions for use.
  • RT-PCR method detects the representative figure of the efficiency of knockdown of EFR3a gene in N2 cells (left side) and normalized quantitative analysis (middle), and the right side shows that knockdown of EFR3a gene does not affect N2a cells. Endocytosis of extracellular A ⁇ 42 .
  • the sequence used to construct the knockdown Efra RNAi was: 5'-AGGTATCATTCAGGTTCTGTT-3' (SEQ ID NO: 4).
  • Example 8 Preparation of PI4KIII ⁇ inhibitor PAO, A1 and G1 solutions and their toxicity test
  • HEK239 cells When testing the toxicity of PAO and A1 on HEK239 cells, HEK239 cells were cultured in DMEM medium containing 50, 100, 150, 200, 250, 300, 400 and 600 nM PAO or A1 for 12 hours, and found to be 250 nM and above by MTT assay. Concentrations of both PAO and A1 kill most cells, and concentrations of 150 nM and below do not. Therefore, 25, 50, 100, and 150 nM PAO and A1 concentrations were selected for culture.
  • PAO powder was dissolved in DMSO to prepare a mother liquor of 30 mg/ml. It was then diluted with distilled water to the desired concentration and the final concentration of DMSO was adjusted to avoid differences in DMSO concentration affecting the experimental results.
  • Three-month-old C57BL/6 mice were first gavaged at doses of 18, 10, and 6 mg/kg body weight, and two mice were intragastrically administered per dose. On day 2, all mice were found dead. Then, the rats were intragastrically administered at a dose of 4.5 and 2.0 mg/kg body weight, and 5 mice were intragastrically administered per dose. For the 4.5 mg/kg body weight dose, one per day was administered, and after 5 consecutive days, 4 out of 5 mice survived.
  • the patient was intragastrically administered once a week from week 1 to week 5 and stopped at the weekend. After 2 consecutive weeks, 5 mice were found to be alive.
  • the median lethal dose of PAO is 2-6 mg/kg body weight, about 4 mg/kg body weight. Therefore, we selected 0.1, 0.3, and 1.0 mg/kg body weight to administer APP/PS1 and control mice, and administrated once a week from week 1 to week 5, and discontinued on weekends for 6 weeks.
  • Example 9 Down-regulation of RBO/PI4KIII ⁇ promotes secretion of A ⁇ 42 by larval tissue culture expressing A ⁇
  • the third instar larvae of Drosophila were sterilized with water and disinfected with 70% alcohol for 2 minutes, cut along the midline of the larvae in Schneider's (Sigma) medium, and the trachea, viscera and adipose tissue of the larvae were carefully removed.
  • the dissected larvae were washed in Schneider's medium and transferred to a 2 ml centrifuge tube containing 150 ul of Schneider's medium and gentamicin (20 mg/ml). There were 5 dissected larvae in each tube and the tubes were placed in a humid, dark, constant temperature environment at 25 °C for 8 hours. Then, 100 ul of each tube was used to quantitatively test A ⁇ 42 by ELISA.
  • the A ⁇ 42 Human ELISA Kit (invitrogen) was used for the ELISA.
  • Fig. 4(a)-(c) show normalized quantitative analysis of different PAO concentration treatments, rbo gene and PI4KIII ⁇ gene mutations in cultured anatomically expressed A ⁇ 42 in medium expressing A ⁇ arc Drosophila third instar larvae. The impact of the level.
  • Example 10 Detection of down-regulation of RBO/PI4KIII ⁇ promotes secretion of A ⁇ 42 by HEK293T cell culture expressing human APP
  • HEK293T cell culture expressing human APP
  • HEK293T cells stably transfected with human APP were cultured in DMEM medium (Hyclone), and 10% FBS (Gibco), penicillin and streptomycin, and G418 (100 ⁇ g/ml) were added.
  • HEK293T cells stably transfected with APP in a 12-well plate containing PAO in the culture solution
  • the concentration was 0, 25, 50, 100 or 150 nM, and after 6-8 hours of culture, equal amounts of cells were collected, respectively.
  • cells were separately lysed with 500 ⁇ l of TBS buffer, centrifuged at low temperature (13,000 g) for 15 minutes, the supernatant was retained, and the pellet was resuspended in 500 ⁇ l of TBS buffer.
  • a 2-fold reaction solution 50 mM Tris-HCl, pH 6.8, 4 mM EDTA, 0.5% CHAPS (w/v)
  • a specific fluorogenic substrate Calbiochem Cat. No. 565764
  • the collected cells were lysed with a protease inhibitor (1% cocktail, invitrogen) TBS buffer, and then immunoblotted with an anti-APP/A ⁇ antibody (6E10).
  • a protease inhibitor 1% cocktail, invitrogen
  • Figure 14 (b) shows immunoblotting A representative graph showing the amount of APP expression of HEK293T cells treated with different PAO concentrations, which was repeated 3 or more.
  • a ⁇ ⁇ -amyloid precursor protein (APP) 42 detects a stably overexpressing secretion of A ⁇ HEK293T cells 42 of the human APP.
  • FIG. 4 (d) - (g) the normalized quantitative analysis of different concentrations A1 and PAO, EFR3a PI4KA knockdown and culture medium human APP stably transfected HEK293 cells the level of A ⁇ 42 influences.
  • PAO treatment produced similar effects in increasing the concentration of A ⁇ 42 in the medium (Fig. 4(d)), and PAO improved culture even in the presence of the ⁇ -secretase inhibitor DAPT (1uM).
  • the concentration of A ⁇ 42 in the base Fig.
  • PAO promoted the secretion of A ⁇ 42 by HEK293 cells stably transfected with APP, but did not change the activity of ⁇ , ⁇ , and ⁇ secretase of splicing APP.
  • Figure 14 (a) did not cause an increase in APP levels.
  • Figure 4 (b) did not cause an increase in APP levels.
  • Example 11 Viral construction packaging and microinjection in mice
  • This most potent miRNA vector is recombined with pDONRTM221 and pLenti6/V5DEST
  • the recombinant reaction yielded the pLENT6/V5-GW/ ⁇ EmGFP-miRNA vector.
  • Lentiviruses were obtained by co-transfection of pLENT6/V5-GW/ ⁇ EmGFP-miRNA vector and Packaging Mix. Viral concentrations were obtained by serial dilution in HEK293T cells. EGFP positive cells were then counted every three days. Silencing efficiency was further obtained by lentiviral transfection of primary cultured hippocampal neurons.
  • APP/PS1 transgenic male rats B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/Mmjax (MMRRC ID 034832-JAX) were cross-bred by mouse hybridization with F1 band (C57BL/6 and C3H).
  • mice were anesthetized with 100 mg/kg Ketamine plus 20 mg/kg Xylazine, fixed on a stereotactic device, and placed on the electric blanket on the abdomen. The head hair was removed, the skin was cut, and a small hole was drilled through the skull.
  • the pump (Harvard Apparatus) was injected through a cannula system (external diameter, 0.29 mm, internal diameter, 0.1 mm, RWD Life Science Co., Ltd.) into 2 ⁇ l of lentivirus solution (virus concentration: 6x10 -7 ) to 2.1 in 20 minutes. Mm posterior to bregma, 2.3mm lateral and 1.9mm ventral. 5 minutes after injection, the needle was removed, the skin was sutured, and the mice were moved to an environment containing sufficient food and water at 25 ° C. At 12 months of age, the anesthesia was small again. Rats were perfused with 4% paraformaldehyde (PFA) in PBS. Experiments were performed on mice following the American Society of Neuroscience policy on animal use.
  • PFA paraformaldehyde
  • Example 12 Detection of knockdown of EFR3a gene by GFP staining of mouse brain slices can repair dendritic atrophy of APP/PS1 mouse neurons
  • Brain slices were blocked with PBS-0.3% triton-5% BSA for 1 hour, and incubated overnight at 4 ° C using rabbit-derived anti-GFP antibody (A11122, invitrogen, 1:100 dilution). It was then washed with PBS and incubated overnight at 4 °C with biotinylated sheep-derived anti-rabbit IgG antibody (H+L) (AbboMax, Inc, 1:100 dilution). The cells were washed again with PBS, and incubated with Cy3-Streptavidin (Jackson ImmunoResearch Laboratories Inc, 1:1000 dilution) for 2 hours at room temperature.
  • the RT-PCR method detects knockdown of the intrinsic EFR3a gene (a) and PI4KA gene (b) in HEK293 cells, knockdown of the overexpressed mouse EFR3a-gfp recombinant gene (c) in HEK293 cells, and The efficiency of the mouse internal EFR3a gene (d) was knocked down in mouse primary cultured hippocampal neurons.
  • the representative figure is on the top, and the normalized method is quantitatively analyzed.
  • confocal imaging showed a full-screen (upper) hippocampal slice of anti-GFP immunostaining and a pyramidal cell (lower) of the CA3 region transfected with lentivirus.
  • a fragment of the dendritic segment (between the two arrows) of about 30 ⁇ m in length was selected to quantify the diameter of the dendrite and the density of the dendritic spines.
  • the scale bars are 500 microns (top) and 50 microns (bottom).
  • EFR3a gene Mouse and humans have two rbo homologous genes, EFR3a gene and EFR3b gene, which are enriched in the AD susceptible region such as hippocampus (Allen brain atlas).
  • AD susceptible region such as hippocampus (Allen brain atlas).
  • Example 13 Analysis by detecting PAO CSF and brain membrane fractions isolated from mouse improvement APP / PS1 mice memory and learning, the increase in CSF A ⁇ 42, A ⁇ 42 but reduced in brain membranes
  • mice were anesthetized with ketamine and xylazine, and the adapter was used to protect the head of the mouse.
  • the hair of the mouse neck was shaved and the skin was cut, and the underlying subcutaneous tissue and muscle were separated to the sides with forceps to expose the portion of the dura mater covering the cisterna magna.
  • the capillary is successfully inserted into the cisterna magna, and the cerebrospinal fluid is drawn into the capillary, collecting approximately 10-20 ⁇ l.
  • the collected cerebrospinal fluid was transferred to a microcentrifuge tube and stored at -80 ° C until use.
  • the detergent-soluble A ⁇ 42 was obtained by a series of extractions, and 5 times the volume of the cerebral hemisphere corresponding to the mouse cerebral hemisphere was ground with tromethamine buffer (TBS), ground to homogenate, and then placed in a centrifuge. The supernatant was centrifuged at 100,000 g for 60 minutes at 4 ° C, and the supernatant was a TBS extract. The lower precipitate was collected, and 5 volumes of TBS buffer containing 1% polyethylene glycol octylphenyl ether was added, ground again and centrifuged, and the upper layer was TBS-Triton extract.
  • TBS tromethamine buffer
  • the lower layer of the precipitate was collected, and 5 volumes of TBS buffer containing 1% SDS was added for a third grinding and centrifugation, and the supernatant was a TBS-SDS extract. Collect the last three clear liquids separately, put them in the refrigerator at -80 °C, and store them for ELISA.
  • Fig. 6(a) the water maze experimental training curves of APP/PS1 mice (left panel) treated with different concentrations of PAO and wild-type mice born in littermates (right panel). For the convenience of comparison, the learning curve of APP/PS1 mice with a PAO concentration of 0 was shown in both the left and right images.
  • Figure 6(b) the search time of the post-training control and APP/PS1 mice in the target quadrant as a percentage of the total search time.
  • the ELISA method was used to quantitatively analyze the cerebrospinal fluid of APP/PS1 mice treated with different concentrations (see Figure (c)), and the brain cell membrane fraction extracted by TBS buffer of 1% Triton and 1% SDS (see Figure 6(d)). A ⁇ 42 levels.
  • the ELISA method was used to quantify the A ⁇ 42 assay in the cerebral cell membrane fraction extracted from the TBS buffer of APP/PS1 mice (left panel) and 1% SDS at 100 °C for different time. influences.
  • the ELISA method quantitatively analyzes A ⁇ 42 levels in brain cell membrane fractions extracted from TBS buffer of 1% Triton and 1% SDS after 60 minutes of treatment at 100 °C.
  • the amount of A? 42 released from each brain cell membrane fraction extracted from 1% Triton and 1% SDS TBS buffer after 100 minutes of treatment at 100 °C accounted for a percentage of the total amount.
  • P values were analyzed by one-way ANOVA.
  • Example 14 Detection of mutant mouse PI4KIII ⁇ by water maze test improves learning and memory ability of APP/PS1 mice
  • Example 15 Effect of PI, PI 4 P, PI 4,5 P on the polymerization of A ⁇ 42 in liposomes by liposome assay
  • a ⁇ 42 and various lipids were mixed together at a ratio of Table 1, and then the organic solvent in the mixture was drained with a freeze dryer.
  • the effects and comparisons of PI, PI 4 P, PI 4 , 5 P on the formation of A ⁇ 42 oligomers in liposomes were analyzed.
  • the left column of Figure 13 shows the PI 4 P versus A ⁇ 42 oligos in liposomes.
  • the promotion of polymer formation is concentration-dependent, and the upper and lower parts are the result of relatively short and long-term post-exposure development of the same immunoblotting membrane; please note the promotion of A ⁇ 42 oligomer formation at a concentration of 80 ⁇ M PI 4 P The effect is weaker than 40 ⁇ M.
  • the right column of Figure 13 shows the effect of PI, PI 4 P and PI 4,5 P on the polymerization of A ⁇ 42 in liposomes, and the upper and lower parts are the result of relatively short and long-term post-exposure development of the same immunoblotting film. It is noted that the effect of PI 4 P is significantly better than that of PI and PI 4,5 P, and that PI and PI 4 P promote the formation of A ⁇ 42 trimer and above oligomers more strongly than PI 4,5 P.
  • Example 16 RBO/EFR3/EFR3A/EFR3B, PI4KIIIa and TTC7 form a complex on the cell membrane
  • yeast EFR3 protein and PI4KIIIa and a scaffolding protein YPP1 form a complex on the cell membrane, and clustered (PIK patchs), The PI 4 P levels of the membrane, even the PI 4, 5 P levels, are controlled together.
  • YPP1 interacts directly with the N-terminal and intermediate regions of the yeast PI4KIIIa protein and plays a key role in the construction and stabilization of PIK patchs (Baird D, Stefan C, et al., 2008, J Cell Biol).
  • composition and function of PIK patchs are also conserved in mammalian cells (Nakatsu F, Baskin JF, et al., 2012, J Cell Biol).
  • the Drosophila homologous protein of TTC7 in Drosophila is encoded by the lethal(2)k14710[(l(2)k14710) gene.
  • transpon-mediated transgenes were introduced into A ⁇ arc Drosophila, one for P ⁇ lacW ⁇ l(2). ) k14710k 15603 , (Bloomington Cat. #11134)
  • the transposon is inserted in the first exon of the (l) k14710 gene, preventing transcription of the (l) k14710 gene; the other is P ⁇ EPgy2 ⁇ bin3 EY09582 , (Bloomington Cat. #20043).
  • This experiment constructed a total of 4 groups of fruit flies: control fruit fly (ctrl), A ⁇ arc fruit fly, A ⁇ containing one copy of P ⁇ lacW ⁇ l(2)k14710k 15603 Arc flies (A ⁇ arc -dttc7 +/- , TTC7 down-regulated) and A ⁇ arc fruit flies (A ⁇ arc -dttc7-OE, TTC7 overexpressing) containing a copy of P ⁇ EPgy2 ⁇ bin3 EY09582 .
  • control fruit fly ctrl
  • a ⁇ arc fruit fly A ⁇ containing one copy of P ⁇ lacW ⁇ l(2)k14710k 15603
  • Arc flies A ⁇ arc -dttc7 +/- , TTC7 down-regulated
  • a ⁇ arc fruit flies A ⁇ arc -dttc7-OE, TTC7 overexpressing
  • FIG 15 shows the effect of down-regulation and overexpression of TTC7 on neurotransmission in A ⁇ arc Drosophila, respectively.
  • Drosophila ctrl
  • overexpressing TTC7 A ⁇ arc-dttc7-OE
  • down-regulating TTC7 expression A ⁇ arc-dttc7+/-

Abstract

本发明提供了一种利用RBO/EFR3/EFR3A/EFR3B抑制剂、TTC7抑制剂以及PI4KIIIα抑制剂来治疗阿尔茨海默病的方法。进一步，本发明还提供了一种以是否促进神经细胞的Aβ分泌来筛选治疗阿尔茨海默病药物的方法。

Description

PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用
关于资助研究的声明
本发明是在国家科技部973项目(2013CB530900)和国家自然基金委面上项目(81371400和81071026)资助下完成。
技术领域
本发明属于医药领域，涉及利用相关抑制剂来下调PI4KIIIα激酶及其与RBO/EFR3/EFR3A/EFR3B蛋白和TTC7蛋白形成的膜蛋白复合体从而治疗阿尔茨海默病的方法；另一方面，本发明涉及根据是否促进细胞的Aβ分泌来筛选用于治疗阿尔茨海默病的药物及治疗靶点的方法。
背景技术
阿尔茨海默病(Alzheimer's disease,AD)是老年人中最常见的神经退行性疾病，以进行性学习记忆能力丧失为特征。突触功能异常和丢失在AD早期发生，被广泛认为是AD学习记忆障碍的一种主要细胞机制，Aβ(尤其是Aβ42)的积累在其中起着重要作用。然而，尽管对大量的旨在抑制Aβ产生和促进其清除的许多化合物或分子进行了临床试验，但还未发现一种化合物或分子可以改善学习记忆或阻止学习记忆障碍进一步恶化。一种可能的解释是这些药物或治疗方法没有改变导致AD学习记忆障碍的关键病理变化，对Aβ在AD中积累的各个方面的认识还待深入。
Aβ不仅在细胞外，也在神经元内积累。大量的证据提示Aβ在神经元的多种细胞内器官中积累，且参与了突触障碍、淀粉样斑块形成、神经元死亡等AD病理变化。另外，被认为对突触和认知功能最具破坏力的寡聚状Aβ在细胞内形成并在AD病人和APP转基因小鼠的大脑神经元内积累或细胞膜中积累。这些膜偶联的Aβ可能扎住在神经元的细胞膜或胞内的细胞器官膜上。导致积神经元Aβ积累的途径可能有多种，比如胞外Aβ内吞、胞内产生的Aβ分泌减少而滞留、自噬泡产生并积累Aβ、胞内Aβ清除下降。
尽管Aβ在细胞内外积累，但AD病人和处于AD早期的病人的脑脊液Aβ42浓度是减低的，约为对照人群的一半。在AD模型小鼠中，脑脊液和脑组织液的Aβ42浓度也呈月龄依赖性减低，脑组织液中也检测不到Aβ二聚体。人们推测脑脊液和脑组织液的Aβ42浓度减低可能是由于胞外淀粉样斑块摄取、Aβ42分泌减少、Aβ42在神经元或大脑细胞膜中积累所致。
已有研究结果揭示，磷脂通过与Aβ相互作用或通过多种细胞和分子过程参与AD病理变化，包括：1)Aβ42能***脂质膜与酸性磷脂相结合，结合的酸性磷脂则促使Aβ42由无序向β折叠的构型转变，从而导致Aβ42在脂质膜上或膜内聚合；2)培养细胞的细胞膜上的4,5二磷酸磷脂酰肌醇(phosphatidylinositol 4,5-phosphate，PIP2)水平与细胞分泌Aβ42的量呈负相关；3)Aβ42表达引起的果蝇神经元功能障碍和学习记忆障碍可通过抑制PI3K而被修复。
在果蝇中rolling blackout(rbo)基因编码一个与二酯酰甘油裂解酶具有一定同源性的膜蛋白RBO。RBO蛋白在果蝇的磷脂代谢、视觉细胞的光传导、突触传递和散装的内吞(bulk endocytosis)中起作用。RBO蛋白从酵母到人都保守。RBO在酵母中的同源蛋白(EFR3)和小鼠的两个同源蛋白(EFR3A和EFR3B)在细胞膜上与IIIα型磷脂酰肌醇4激酶(PI4KIIIα)和一个脚手架蛋白(在哺乳动物中称为 Tetratricopeptide repeat domain 7，TTC7，参见Baird D,Stefan C,et al.,2008,J Cell Biol；Nakatsu F,Baskin JF,et al.,2012,J Cell Biol)形成复合体，从而将PI4KIIIα锚泊在细胞膜上，并进一步调控细胞膜上4磷酸磷脂酰肌醇(phosphatidylinositol 4-phosphate，PI4P)和PIP2的水平。
在果蝇中，泛神经元表达与分泌信号肽融合的Aβ42导致神经元内Aβ42积累和神经退行性改变。之前，发明人在成虫果蝇的一个简单神经通路，即巨纤维通路(giant fiber pathway，GF)中表达含有分泌信号肽的Aβ42，发现Aβ42在神经元内积累，并导致了日龄依赖(age-dependent)的突出传递失败和运动能力下降等障碍。这种表达Aβ42的果蝇为研究一些可能与AD相关的基因在神经元内Aβ42积累及其引起的突触障碍中的作用提供了很好的平台。有鉴于此，发明人利用该模型检测了基因rbo、PI4KIIIα及ttc7的突变或过量表达，以及常用的PI4KIIIα蛋白抑制剂对该模型中神经退行性病变的影响。发明人还在APP/PS1转基因小鼠中，研究了敲减rbo基因的一个小鼠同源基因Efr3a对海马神经元萎缩，以及利用常见的PI4KIIIα激酶的小分子抑制剂PAO灌胃对学习记忆、脑脊液中和大脑实质的细胞膜组分中Aβ42浓度的影响，以及下调PI4KA基因(编码PI4KIIIα蛋白)对学习记忆的影响，以及PI4KIIIα的产物PI4P对Aβ42在脂质体中形成聚合体的影响。
发明内容
本发明公开了利用遗传手段或相关抑制剂抑制下调PI4KIIIα蛋白、RBO/EFR3/EFR3A/EFR3B膜蛋白、TTC7蛋白、以及它们形成的膜蛋白复合体的数量或其相关的酶活性能够促进神经元细胞的Aβ(尤其是Aβ42)分泌并相应减少了神经元内的Aβ积累，从而可以减轻AD模型果蝇和小鼠的神经功能障碍。本发明因此揭示了神经元的Aβ42分泌在AD治疗中的重要作用，为治疗AD提供了全新的策略；同时，本发明也提供了治疗AD的新药物，并为进一步筛选治疗AD的药物和治疗靶 点指明了新方向。
一方面，本发明涉及新的治疗阿尔茨海默病的方法。根据本发明的一个具体实施例，本发明公开了一种利用PI4KIIIα抑制剂治疗阿尔茨海默病的方法，所述PI4KIIIα抑制剂可以为抗PI4KIIIα抗体、对PI4KIIIα特异的抑制性核苷酸、或对PI4KIIIα特异的小分子化合物抑制剂。优选地，所述对PI4KIIIα特异的抑制性核苷酸可以为如SEQ ID NO：6所示的核苷酸序列；所述PI4KIIIα抑制剂选自下列小分子化合物抑制剂中的一种或多种：PAO、PAO的衍生物、G1、A1、G1或A1的类似物。
根据本发明的另一具体实施例，本发明还公开了一种利用RBO/EFR3/EFR3A/EFR3B抑制剂治疗阿尔茨海默病的方法。所述RBO/EFR3/EFR3A/EFR3B抑制剂可以为抗RBO/EFR3/EFR3A/EFR3B抗体。
同时，根据本发明的另一具体实施例，本发明还公开了一种利用PI4P抑制剂治疗阿尔茨海默病的方法。所述PI4P抑制剂可以为抗PI4P抗体、OSH2-PH2X融合蛋白、或者OSH2-2x-PH融合蛋白。
另一方面，本发明还涉及一种可以用来治疗阿尔茨海默病的药物组合物，其包含下列物质的一种或多种：PI4KIIIα抑制剂、RBO/EFR3/EFR3A/EFR3B抑制剂以及PI4P抑制剂，以及任选的药物载体。优选地，该药物组合物还可包含一种或多种抗Aβ抗体和/或能清除神经细胞外Aβ聚合物的化合物，所述能清除神经细胞外Aβ聚合物的化合物选自：海洋硫酸寡糖类HSH971及其类似物、阿坎酸及其类似物、以及依达拉奉及其类似物。
此外，本发明还涉及如何筛选治疗阿尔茨海默病的药物的方法。根据本发明的一个具体实施例，本发明公开了一种以PI4KIIIα蛋白的 激酶活性为治疗靶点的筛选阿尔茨海默病药物的方法，所述方法包括以下步骤：观察候选药物对PI4KIIIα蛋白的磷酸激酶活性的影响，若候选药物能够抑制PI4KIIIα的磷酸激酶活性，则表明该候选药物为治疗阿尔茨海默病的潜在药物。
根据本发明的另一具体实施例，本发明同时也公开了一种以RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白以及PI4KIIIα蛋白的相互作用为治疗靶点的筛选阿尔茨海默病药物的方法，包括以下步骤：观察候选药物对RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白以及PI4KIIIα蛋白的相互作用的影响，若候选药物能够抑制RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白和PI4KIIIα蛋白的相互作用，从而减少RBO/EFR3/EFR3A/EFR3B-TTC7-PI4KIIIα蛋白复合体的形成，则表明该候选药物是治疗阿尔茨海默病的潜在药物。
根据本发明的另一具体实施例，本发明同时还公开了一种以细胞膜上的PI4P的水平为治疗靶点的筛选阿尔茨海默病药物的方法，所述方法包括以下步骤：观察候选药物是否对细胞膜上的PI4P水平有影响，若候选药物能够抑制细胞膜上的PI4P水平，则表明该候选药物是治疗阿尔茨海默病的潜在药物。
附图说明
图1示出rbo基因突变缓解表达Aβarc的果蝇的神经功能损伤，减低PI4KIIIα蛋白的表达量或减弱了PI4KIIIα蛋白与RBO蛋白的相互作用。
图2示出下调PI4KIIIα蛋白表达量或药物抑制PI4KIIIα酶活性缓解表达Aβarc的果蝇的神经功能损伤。
图3示出下调RBO/PI4KIIIα蛋白表达量或功能降低神经元内Aβ的积累。
图4示出下调RBO/PI4KIIIα蛋白表达量或功能促进Aβ42的分泌。
图5示出在APP/PS1和对照小鼠中敲减Efr3a基因对海马CA3和 DG区神经元的树突的直径和树突棘密度的影响。
图6示出PAO处理对APP/PS1小鼠和对照小鼠的学***的影响。
图7示出rbo基因突变缓解表达Aβ42的果蝇的神经功能损伤。
图8示出rbo基因和shibire基因突变分别对过表达Aβarc或果蝇Tau蛋白的果蝇的运动能力和寿命的影响。
图9示出rboS358A基因和itprSV35基因突变对表达Aβarc果蝇的影响。
图10示出敲减Efr3a基因对N2a细胞内吞细胞外Aβ42的影响和rbo基因及PI4KA基因突变对Aβarc表达果蝇中Aβarc转录的影响。
图11示出在HEK293细胞或小鼠原代培养的海马神经元中敲减Efr3a基因或PI4KA基因的效率。
图12示在APP/PS1小鼠的一个拷贝PI4KA基因中***转座子明显改善小鼠的学习和记忆能力。
图13示PI4P促进Aβ42在脂质体中形成寡聚体。
图14示PAO对APP表达量和α,β,γ分泌酶活性的影响。
图15示ttc7基因突变和过量表达对Aβarc果蝇的神经功能损伤的影响。
具体实施方式
本发明中，包括说明书和权利要求书，除非特别说明，使用的下列术语具有如下的含义：
本发明所用的术语“rbo/Efr3/Efr3a/Efr3b基因”是指源自果蝇的rbo基因、源自酵母的Efr3、或源自哺乳动物Efr3a基因以及Efr3b基因；本发明所用术语“RBO/EFR3/EFR3A/EFR3B蛋白”是指源自果蝇的rbo基因、源自酵母的Efr3基因、或源自哺乳动物的Efr3a/Efr3b基因所编码的蛋白。
本发明所用的术语“PI4KIIIα/PI4KA基因”是指源自果蝇或哺乳 动物的PI4KIIIα基因或PI4KA基因；本发明所用术语“PI4KIIIα蛋白”是指果蝇或哺乳动物中由PI4KIIIα/PI4KA基因所编码的蛋白。
本发明所用的术语“ttc7基因”是指源自哺乳动物的ttc7基因；本发明所用术语“TTC7蛋白”是指前述哺乳动物中由ttc7基因所编码的蛋白。
本发明所用的术语“抑制剂”是指能够起到降低、减少、或消除目标物的数量、特定功能、以及特定性质的物质。所述目标物可以为蛋白、多肽、核酸等，而所述抑制剂以所述目标物的数量、特定功能以及特定性质为直接或间接的作用对象，可以使得所述目标物的数量、特定功能以及特定性质产生相应的降低、减少或消除。所述抑制剂可以为蛋白、多肽、核酸、小分子化合物等。
例如，本发明所用的术语“PI4KIIIα抑制剂”是指能够降低、减少、消除PI4KIIIα/PI4KA基因的表达、转录、翻译，和/或其形成的PI4KIIIα蛋白的稳定性、与膜蛋白RBO\EFR3\EFR3A\EFR3B以及TTC7蛋白的结合能力、以及具有的磷酸激酶活性等各方面的各类物质，包括但不限于对PI4KIIIα/PI4KA基因特异的抑制性核苷酸、抗PI4KIIIα蛋白抗体、能够抑制PI4KIIIα激酶活性的小分子化合物，和/或能够抑制PI4KIIIα蛋白与其他膜蛋白相互作用的各种物质等。
类似地，本发明所用的术语“RBO/EFR3/EFR3A/EFR3B抑制剂”是指能够抑制、降低、消除rbo/Efr3/Efr3a/Efr3b基因的表达、转录、翻译、和/或其形成的RBO/EFR3/EFR3A/EFR3B蛋白的稳定性、其与PI4KIIIα蛋白的结合能力等各方面的各类物质，包括但不限于对rbo/Efr3/Efr3a/Efr3b基因特异的抑制性核苷酸、抗RBO/EFR3/EFR3A/EFR3B蛋白的抗体、以及能够抑制RBO/EFR3/EFR3A/EFR3B蛋白与PI4KIIIα蛋白形成复合体的各种物质。
本发明所用的术语“TTC7抑制剂”是指能够降低、减少、消除ttc7基因的表达、转录、翻译、和/或其形成的TTC7蛋白的稳定性、与膜蛋白RBO\EFR3\EFR3A\EFR3B的结合能力等各方面的各类物质，包括但不限于对ttc7基因特异的抑制性核苷酸、抗TTC7蛋白抗体、和/或能够抑制TTC7蛋白与膜蛋白RBO\EFR3\EFR3A\EFR3B相互作用的各种物质等。
同样，本发明所用术语“PI4P抑制剂”是指能够抑制、降低、消除细胞膜上PI4P数量水平的各类物质，包括但不限于抗PI4P抗体、以及能与PI4P特异结合的OSH2-PH2X融合蛋白或者OSH2-2x-PH融合蛋白等。
本发明所用的术语“抗体”，是指结合特定表位的任何免疫球蛋白或完整分子以及其片段。所述抗体包括但不限于多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体、单链抗体、以及完整抗体的片段和/或部分，只要这些片段或部分保留亲本抗体的抗原结合能力。例如，本发明中，“抗PI4KIIIα抗体”是指能特异性地结合PI4KIIIα蛋白，或其功能变体或功能片段的单克隆抗体、多克隆抗体、单链抗体和其具有免疫活性的片段或部分。本发明中，诸如“PI4KIIIα抗体”、“抗PI4KIIIα抗体”以及“针对PI4KIIIα的抗体”这样的术语均可以互换使用。
本发明中，“功能变体”是指本发明的蛋白或多肽，其氨基酸序列上一个或多个氨基酸发生了改变。所述改变可以是“保守性”改变(其中取代的氨基酸具有相似结构或化学特性)也可以是“非保守性”变化；类似的变化还可包括氨基酸缺失或***或两者均有。但这些氨基酸残基的改变或氨基酸的缺失或***，均不应实质性地改变或破坏原氨基酸序列的生物学或免疫学活性和功能。类似地，本发明中，“功能片段”是指本发明的蛋白或多肽的任何部分，所述部分保留其作为一部分的蛋白或多肽(即“亲本”蛋白或多肽)的基本相似或相同的 生物学或免疫学活性和功能。
本发明所用的术语“抑制性核苷酸”是指能够结合并抑制特定基因表达的核苷酸化合物。典型的抑制性核苷酸包括但不限于反义寡核苷酸(antisense oligonucleotides)、三链螺旋DNA(triple helix DNA)、RNA适配体(aptamers)、核酶(ribozymes)、短抑制核糖核苷酸(siRNA)、短发夹RNA(shRNA)和microRNA。这些核苷酸化合物能以比对于其他核苷酸序列更大的亲和力与所述特定基因相结合，从而抑制特定基因的表达。
本发明所用术语“小分子化合物”是指一种分子量小于3千道尔顿的有机化合物，该有机化合物可以是天然的或者是化学合成的。本发明所用术语“衍生物”是指通过一种或多种化学反应对母体有机化合物进行修饰所产生的化合物，其与母体有机化合物具有相似的结构，在功能上具有相似的效果。本发明所用术语“类似物”则是指这样一类有机化合物，其并不一定是通过对母体有机化合物进行化学修饰而获得的，但其从结构上看与母体有机化合物相似，且在功能上也具有相似的效果。
本发明所用术语“阿尔茨海默病”(AD)是指一种以进行性学习记忆障碍为突出临床症状的老年性神经退行性疾病。大部分AD患者在中晚期有神经细胞外beta淀粉样斑块，细胞内有Tau蛋白组成的神经纤维缠结、或突触及神经细胞丢失。该疾病既可存在于人，也可存在于动物，比如狗。
本发明所用术语“Aβ”是指与由淀粉样前体蛋白(Amyloid Precursor Protein，APP)经分泌酶剪切而产生的一系列长度在38-48个氨基酸之间的多肽，主要是Aβ38、Aβ40、Aβ42、Aβ44和Aβ45等氨基酸序列相同的多肽。本发明中，利用特定的表达***，Aβ也可以通过利用转基因或病毒载体感染细胞等方法表达的Aβ融合蛋白经其它蛋白剪切酶剪 切而来，比如Aβ的N端可以与来源于果蝇necrotic基因编码的蛋白的分泌信号肽(氨基酸序列：MASKVSILLLLTVHLLAAQTFAQDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA)(SEQ ID NO：1)或来源于大鼠Proenkephalin前体(pre-proenkephalin)的分泌信号肽(氨基酸序列：MAQFLRLCIWLLALGSCLLATVQA(SEQ ID NO：2))等组成的融合蛋白。
本发明所用术语“Aβ分泌”是指Aβ在细胞内或细胞膜产生后经由细胞膜排出到胞外的过程，该过程可导致细胞内的Aβ积累减少。其中，“Aβ42分泌”是专门指Aβ42在细胞内或细胞膜产生后经由细胞膜排出到胞外的过程，该过程可导致细胞内的Aβ42积累减少。
本发明所用术语“治疗靶点”是指可用于治疗某种疾病的各种物质及其在动物体或人体的作用目标。所述物质作用于所述作用目标可以起到治疗该种疾病的效果。所述物质可以为蛋白、多肽、核酸、小分子化合物等各类物质，所述作用目标可以是某个基因(包括基因的特定序列)、某个蛋白(包括蛋白的某个特定位点)、某种蛋白复合体(包括其特定的结合部位)等这样的物质实体，也可以是前述基因和/或蛋白的某种特性、某种功能、某种与周边物质、环境的相互关系等，只要所述物质能够影响该基因、蛋白、蛋白复合体，或其特定的特性、功能、相互关系等，从而能够起到治疗该疾病的作用。
本发明所用的术语“治疗”是指逆转、减轻或抑制该术语所应用的疾病的进展，或疾病的一种或多种症状。如本文所使用的，根据患者的状况，该术语也包括预防疾病，包括预防疾病或与其相关的任何症状的发作，以及减轻病症或其在发作前的任何病状的严重性。
本发明所用术语“抑制”、“削弱”、“下调”、“消除”等均是指数量或程度上的减少或减轻。这种减少或减轻并不限于任何幅度，只要表现出这种趋势即可。例如，该种减少或减轻相对于原来的数量 或程度可以是100％，也可以是50％，也可以是1％甚至更少。
本发明揭示了下调RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白或PI4KIIIα蛋白的表达、削弱RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白、与PI4KIIIα蛋白之间的相互作用、以及抑制PI4KIIIα的酶活性等一系列行为均可改善表达Aβ42的神经细胞随着年龄的增长而出现的突触功能减退和丢失等缺陷，并且公开了这些作用是通过促进神经细胞的Aβ(尤其是Aβ42)分泌，减少神经细胞膜或神经细胞内的Aβ(尤其是Aβ42)累积所致。
例如，本发明中，发明人发现敲减Efr3a基因可以减少APP/PS1双转基因小鼠的海马神经元树突和棘的萎缩，而用一种常用的PI4KIIIα蛋白抑制剂－氧化苯砷(Phenylarsine Oxide，PAO)进行灌胃则可以明显改善APP/PS1小鼠的学习和记忆能力，减少了脑组织细胞膜偶联的Aβ42尤其是聚合状Aβ42的含量，尽管该过程同时伴随有脑脊液中Aβ42含量增加。这些结果揭示，APP/PS1小鼠的痴呆症状可以通过促进神经元的Aβ42分泌，减少神经元内或神经元细胞膜上Aβ42(特别是聚合状Aβ42)的累积而得到改善。
发明人发现，通过遗传方法适度下调细胞或神经元内RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白、以及PI4KIIIα蛋白的表达，或阻止它们形成蛋白复合物以减少PI4KIIIα蛋白的膜上分布，或抑制PI4KIIIα蛋白的磷酸激酶活性，均能起到促进细胞或神经元的Aβ42分泌，减少神经元内Aβ42积累，缓解AD相关的神经退行性改变以及功能障碍的作用，但同时，无论是Aβ42或APP的表达量，还是剪切APP的α、β和γ分泌酶的活性，均未受到明显影响。发明人另外还发现，PI4KIIIα蛋白的产物PI4P在脂质体中促进Aβ42单体聚合形成多聚体，且该促进作用比PI4P的前体(PI)和其衍生物PI4,5P的促进作用要强很多。
发明人认为，Aβ(包括Aβ42)产自于细胞质膜上或细胞内器官，而产生的Aβ可能通过被动释放、胞吐、溶酶体介导的释放、或其它还未被发现的途径被分泌出细胞。但是，不管Aβ产自于哪儿，如何被分泌，细胞质膜应是Aβ离开细胞必须经过的最后通路。由于Aβ的疏水性，在细胞质膜上，Aβ一方面***疏水的脂肪酸链区，另一方面又与磷脂酰肌醇(尤其是磷酸化磷脂酰肌醇中的PI4P)和其他酸性磷脂相互作用，这种作用促使Aβ从无序构型向β折叠构型转变，进而聚集形成Aβ聚合体沉积于膜上或随后被内吞而在细胞内积累。此外，研究表明，可溶Aβ(包括Aβ42)聚合体与细胞膜或脂质体膜的亲和力要比Aβ单体强许多。因此，形成聚合体的Aβ将更容易累积在细胞膜上。
PI4P是细胞膜上磷酸化磷脂酰肌醇的主要组成成分之一，其对Aβ聚合体形成的促进作用比PI和PI4,5P的作用要强。本发明中，发明人发现，PI4P对处于脂质中的Aβ42形成聚合体的促进作用呈现明显的剂量依赖性。下调RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白或PI4KIIIα蛋白的表达，抑制它们之间形成复合物，或者抑制PI4KIIIα蛋白的激酶功能，均将减少细胞膜上PI4P的生成量。因此，下调RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白和/或PI4KIIIα蛋白的表达，或阻止RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白和PI4KIIIα蛋白形成附着在膜上的蛋白复合体，或抑制PI4KIIIα蛋白相应的磷酸激酶功能，均将实质性地降低细胞质膜上磷酸化磷脂酰肌醇(尤其是PI4P)的含量，从而削弱细胞质膜上Aβ(包括Aβ42)与磷酸化磷脂酰肌醇的相互作用，从而导致细胞质膜上的Aβ更多地以无序的Aβ单体形式存在。如前所述，这种无序形式的Aβ单体和膜的亲和力低，很容易被从膜上释放出来而分泌到细胞外。因此，上述调节行为能在APP表达量和α、β和γ分泌酶活性无明显变化的情况下，有效地减低胞内Aβ的积累，并导致胞外Aβ水平明显增多。
已有研究报导发现，果蝇细胞内Aβ42的累积可激活PI3K和与之相关的PI3K/Akt信号通路，从而诱发AD相关的突触功能损害和长期记 忆缺失；相应地，相关研究认为抑制PI3K的活性也可以作为治疗AD的手段。但本发明中，发明人认为细胞中Aβ42的累积并不是因磷脂酰肌醇激酶(包括PI3K)激活其所相关联的PI3K/Akt信号通路所造成的，而更直接地是因细胞质膜上磷脂酰肌醇激酶被激活后磷酸化膜上的磷脂酰肌醇所导致的结果；此外，本发明所涉及的磷脂酰肌醇激酶主要为PI4K，而并非涉及PI3K/Akt信号通路的PI3K。例如，发明人发现，采用针对PI4K具有较高特异性但对PI3K不敏感的磷脂酰肌醇激酶抑制剂，例如PAO，在较低浓度下即可以有效地促进细胞的Aβ42分泌。
因此，本发明揭示，为了实现治疗AD的目的，可以采用促进Aβ分泌，尤其是促进Aβ42分泌的方式，以减少Aβ(包括Aβ42)在神经细胞内或细胞膜上的累积。但是，增加的Aβ分泌，不能是可归因于上调了APP的表达量，或是改变了α、β和γ分泌酶的活性而导致的Aβ的产生量增多。
本领域普通技术人员了解，促进神经细胞的Aβ分泌可以采用很多种形式，包括削弱Aβ与细胞膜上糖、脂和蛋白的结合或相互作用等。本发明中，优选为如前所述通过减少Aβ(尤其是Aβ42)在细胞膜上形成聚合体的方法来促进细胞的Aβ分泌。
此外，本发明还揭示，由于调节RBO/EFR3/EFR3A/EFR3B蛋白、TTC7蛋白与PI4KIIIα蛋白的数量和它们形成复合物的能力，以及调节PI4KIIIα蛋白的磷酸激酶活性，均可减少Aβ在细胞膜上形成的聚合体，从而促进细胞的Aβ(尤其是Aβ42)分泌，因此这些蛋白以及这些蛋白的相互关系均能构成对AD治疗的潜在治疗靶点。
据此，本领域普通技术人员可以了解，能够抑制、降低、减弱、或者消除rbo/Efr3/Efr3a/Efr3b基因的表达、转录或翻译以及降低其编码的RBO/EFR3/EFR3A/EFR3B蛋白的稳定性的抑制剂和方法，以及能够阻止它和磷酸肌醇激酶PI4KIIIα蛋白以及TTC7蛋白形成蛋白复合 物的抑制剂和方法，均可用于治疗AD。所述RBO/EFR3/EFR3A/EFR3B抑制剂包括但不限于rbo/Efr3/Efr3a/Efr3b基因的抑制性核苷酸(包括反义RNA、siRNA或miRNA等)，以及抗RBO/EFR3/EFR3A/EFR3B蛋白的抗体等。
本领域技术人员了解，rbo/Efr3/Efr3a/Efr3b基因的抑制性核苷酸及其制备方法已经公开(例如，可参见www.genecards.org，以及已有的产品：ORIGENE，Cat.#SR308056和Cat.#TR303768)。类似地，抗RBO/EFR3/EFR3A/EFR3B抗体的制备方法也已经公开(例如，可参考www.genecards.org，以及已有的产品：Novus，Cat.#NBP1-81539；Thermo Fisher Scientific Cat.#PA5-24904)。
此外，如前所述，调节PI4KIIIα/PI4KA基因在细胞上的表达、转录、翻译，调节PI4KIIIα/PI4KA基因编码的PI4KIIIα蛋白的稳定性，调节该蛋白和膜蛋白RBO/EFR3/EFR3A/EFR3B以及TTC7蛋白形成蛋白复合体的能力，以及调节该磷酸激酶的酶活性，也均可作为AD治疗的手段。本领域普通技术人员因此应该知道，能够抑制、降低、减弱、消除PI4KIIIα/PI4KA基因的表达、转录、翻译，或其编码的PI4KIIIα蛋白的稳定性和其生物活性的抑制剂和方法，包括但不限于对PI4KIIIα/PI4KA基因特异的抑制性核苷酸，抗PI4KIIIα蛋白的抗体能够抑制PI4KIIIα蛋白与膜蛋白形成膜蛋白复合体，以及能够抑制激酶活性的小分子化合物抑制剂等，均可用于治疗AD。优选地，该抑制剂为小分子化合物，例如，PAO(氧化苯砷或Phenylarsine Oxide)，PAO的衍生物，A1，G1，或A1以及G1的类似物。更优选地，该抑制剂可以为PAO或其衍生物。
本领域技术人员了解，PAO是一种以氧化砷基与苯环为基本结构的小分子化合物，其对PI4KIIIα蛋白的磷酸激酶活性具有很强的抑制 作用。PAO的化学式结构为：
Figure PCTCN2015078058-appb-000001
苯基氧化砷(氧化苯砷)(oxo(phenyl)arsane)
PAO的制备方法为本领域技术人员所熟知。根据本发明，可以用于治疗AD的还包括PAO的各种衍生物，只要这些衍生物具有抑制PI4KIIIα蛋白的磷酸激酶活性的作用。本领域技术人员了解，该类衍生物的制备方法均已公开。
同样，本领域技术人员了解，A1和G1均为PI4KIIIα蛋白的小分子化合物抑制剂，具有较类似的结构。其中，A1的化合结构式为：
Figure PCTCN2015078058-appb-000002
5-(2-氨基-1-(4-(4-吗啉基)苯基)-1H-苯并咪唑-6-基)-N-(2-氟代苯基)-2-甲氧基-3-吡啶磺酰胺
G1的化学结构式为：
Figure PCTCN2015078058-appb-000003
(aS)-5-(2-氨基-4-氧代-3-(2-(三氟甲基)苯基)-3,4-二氢喹唑啉-6-基)-N-(2,4-二氟代苯基)-2-甲氧基吡啶-3-磺酰胺
A1和G1的制备方法均已公开(例如，可参见Bojjireddy,N.,et al.(2014),JBC 289:6120-6132和Leivers,A.L.,et al.(2014),JMC57:2091-2106)。根据本发明，A1和G1的结构类似物，只要它们具备抑制PI4KIIIα蛋白的磷酸激酶活性的功能，也同样可以用于治疗AD。本领域技术人员了解，该类结构类似物的制备方法也均已公开。
此外，PI4KIIIα蛋白与RBO/EFR3/EFR3A/EFR3B蛋白结合形成膜蛋白复合体需要相应的脚手架蛋白TTC7的协助。因此，根据本发明，本领域普通技术人员可以了解，能够抑制、降低、减弱、或者消除ttc7基因的表达、转录或翻译以及降低其编码的TTC7蛋白的稳定性的抑制剂和方法，以及能够阻止它和RBO/EFR3/EFR3A/EFR3B蛋白以及PI4KIIIα蛋白形成蛋白复合物的抑制剂和方法，均可用于治疗AD。所述TTC7抑制剂包括但不限于ttc7基因的抑制性核苷酸(包括反义RNA、siRNA或miRNA等)，以及抗TTC7蛋白的抗体等。
再者，根据本发明，下调RBO/EFR3/EFR3A/EFR3B蛋白和PI4KIIIα蛋白的表达，或阻止它们形成复合物以减少PI4KIIIα蛋白的膜上分布，或抑制PI4KIIIα蛋白的磷酸激酶活性，实质上均导致的是细胞膜上PI4P减少，进而促进细胞的Aβ分泌。因此，本领域普通技术人员了解，任何时候能减少细胞膜上PI4P数量水平的抑制剂或方法，进而可以减少Aβ在细胞膜上形成多聚体，也均能起到前述治疗AD的作用。
本领域技术人员了解，前述PI4P抑制剂可以是能与PI4P特异结合的抗体或其它分子。目前抗PI4P抗体的制备方法已经公开(参见Brown BK and Karasavass N,et al.,2007,J virol；Wassef NM and Roerdink F,et al.1984,Mol Immuol)。例如，可以是人源的广谱中和抗体4E10和其它抗PI4P抗体。目前与PI4P特异结合的分子的制备方法也已经公开(Balla A and Kim YJ,et al.,2008,Mol Biol Cell；Zubenko GS and Stiffler et al.,1999,Biol Psychiary)。例如，可以是OSH2-PH2X融合蛋白或者OSH2-2x-PH融合蛋白。
本发明所提供的上述各种可以用于治疗AD或具有治疗AD的潜力物质(笼统称为“本发明物质”)，包括但不限于抗RBO/EFR3/EFR3A/EFR3B抗体、抗PI4KIIIα抗体、抗TTC7抗体，抗PI4P抗体、对rbo/Efr3/Efr3a/Efr3b基因特异的抑制性核苷酸、对PI4KIIIα/PI4KA基因特异的抑制性核苷酸、以及对PI4KIIIα蛋白的磷酸激酶作用有抑制作用的小分子化合物等，均可以是分离的、纯化的、合成的、和/或重组的。
此外，本发明物质还可以配制成组合物，诸如药物组合物。在这一点上，本发明提供包括前面所述任意抗体、抑制性核苷酸、或/和小分子化合物等抑制剂，以及相应的药用载体的药物组合物。
包含任意本发明物质的药物组合物可以包括多于一种本发明物质，例如：抗体和小分子化合物、抑制性核苷酸和抗体，或两种或更多不同的抗体或者小分子化合物等。备选地，所述药物组合物还可以包括与另一种或多种具有药物活性的试剂或药物的组合。例如，可以包括抗Aβ的抗体药物，如Bapineuzumab，或者是可以与大脑内神经细胞外的Aβ或β淀粉样斑块结合，阻滞Aβ聚合或促进聚合状的Aβ解聚的化合物，比如海洋硫酸寡糖类HSH971及其类似物、阿坎酸(tramiprosate)及其类似物、依达拉奉(Edaravone)及其类似物等。这样，该药物组合物一方面促进Aβ从神经元内分泌出来，一方面促进神经元外的Aβ清除，从而可达到更好的治疗AD的效果。
另一方面，本发明同时还揭示了一种新的用于筛选治疗AD的药物或治疗靶点的方法。该方法的设计基于前述本发明的新发现，也即采用促进Aβ42分泌的方式可以减少胞内的Aβ42的累积，从而可以减缓和防止AD相关的神经退化和功能障碍。因此，筛选候选药物或治疗靶点的标准是应用该药物或调节该治疗靶点后能够达到促进细胞的Aβ分泌的效果，尤其是Aβ42的分泌，且增加的Aβ分泌不能归因于是因为 上调了APP的表达量，或者改变了α、β或γ分泌酶的活性而导致的Aβ的产生增多。
根据本发明，所述治疗靶点的调节是指利用相关物质对该治疗靶点进行直接或间接的作用，从而导致该治疗靶点的功能、性质或与周围环境的相关关系发生改变，因而能够产生或引发产生相应的促进细胞的Aβ分泌的效果，尤其是Aβ42的分泌。
普通技术人员可以了解，为了选出有效的治疗阿尔茨海默病的药物，可以用于筛选试验的细胞系可为哺乳动物、昆虫等的真核细胞系，比如：HEK293、COS7、N2a、SH-SY5Y、S2和sf9等。所述方法包括检测候选药物是否能减少细胞膜或细胞内的Aβ积累，尤其是Aβ42的积累，从而选出有效的治疗阿尔兹海茨海默病的药物。优选地，所述Aβ42分泌是否增加的检测实验可利用过量表达APP的细胞系(如稳转人源APP的HEK293、COS7、N2a、SH-SY5Y细胞系)来进行，或者利用果蝇模型进行，优选是果蝇三龄幼虫组织。所述Aβ42分泌是否增加可以采用免疫分析方法来检测，包括酶联免疫吸附剂测定方法(enzyme-linked immunosorbent assay，ELISA)或电化学发光免疫分析(Electrochemiluminescence，ECLIA)方法。
优选地，所述用于筛选治疗AD的药物或调节靶点的方法可以包括首先观察候选药物或调节靶点的应用对PI4KIIIα酶活性的影响，若候选药物或调节靶点的应用能够使检测***中PI4KIIIα激酶的作用效果发生负向改变，即表征PI4KIIIα酶活性减弱或细胞膜上PI4P水平下降，则表明该候选药物、试剂或靶点是治疗AD的潜在药物或调节靶点。通过这样的筛选，再检测其是否对细胞内Aβ(尤其是Aβ42)积累造成减少，是否促进Aβ向胞外的分泌。采用这样的方法，可以大大提高候选对象的筛选效率。
根据本发明的一个具体实施例，所述筛选用于治疗AD的药物或 治疗靶点的方法可以通过直接检测候选药物的应用或治疗靶点的调节是否能使原来定位在细胞膜的PI4KIIIα蛋白向胞浆分布，从而减少PI4KIIIα蛋白在细胞膜上的量，导致减少细胞膜上的Aβ单体向聚合体的转变并增加向胞外的分泌来判断。优选地，可以选择观察细胞膜上利用荧光标记的PI4KIIIα，比如荧光蛋白标记的PI4KIIIα(GFP-PI4KIIIα)，观察该荧光标记的PI4KIIIα是否从细胞膜上向胞浆转移。
根据本发明的一个具体实施例，所述筛选用于治疗AD的药物或治疗靶点的方法还可以利用包括免疫共沉淀等检测蛋白与蛋白相互作用的方法来进行。如候选药物的应用或者治疗靶点的调节能够使RBO/EFR3/EFR3A/EFR3B蛋白与TTC7蛋白以及PI4KIIIα蛋白相互作用减弱，即表征该药物或靶点能够削弱PI4KIIIα蛋白形成膜蛋复合体白的能力，从而减少细胞膜上的Aβ单体向聚合体转变，增加向胞外的分泌。
根据本发明的一个具体实施例，所述筛选用于治疗AD的药物或治疗靶点的方法还可以通过直接检测候选药物的应用或者治疗靶点的调节是否能够使细胞膜的PI4P水平下降来实现。优选地，可以利用荧光显微镜、共聚焦或双光子显微镜观察细胞膜上荧光标记的特异结合PI4P的分子，比如荧光蛋白标记的OSH2-PH2X或者OSH2-2x-PH融蛋白，在细胞膜上的量是否减少，或是否从细胞膜上向胞浆转移来判断。
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
以下数据主要是通过动物和细胞培养实验获得的，选择SPSS软件分析。如其他地方未特别标明，数据采用mean±sem表示。P＜0.05为差异有统计学意义。这里和以下显示的所有数据均以mean±sem表示，“*”，“**”和“***”分别表示p<0.05，0.01和0.001。
实施例1：果蝇品系与遗传学方法
标准培养基，在12小时光照12小时黑暗交替循环，恒温25℃条件下培养。
本发明采用了下列转基因果蝇品系：rboS358A、[UAS]Aβarc、[UAS]Aβ42、[UAS]dtau、[UAS]mCD8-gfp(由Dr.Z.Wang提供)、[UAS]shibirets1(由Dr.A Guo提供)、[Gal4]A307(由Dr.O’Kane提供)。其中，rboS358A基因是用含有rbo基因的野生型基因组DNA经定点突变后构建的转基因，其表达由rbo基因本身的前驱动子驱动。[Gal4]A307表达转录因子Gal4，用于驱动[UAS]Aβarc、[UAS]Aβ42、[UAS]dtau、[UAS]mCD8-gfp、[UAS]shibirets1转基因在果蝇的巨纤维通路(Giant Fiber system)神经元和少量其它神经元表达Aβarc、Aβ42、dTau、mCD8-GFP或温度敏感型突变的Dynamin。采用的果蝇突变体有：rbots1(温度敏感的错义突变)、rbo2(敲除突变)、itprsv35(无义突变，Bloomington Stock#30740)、PI4KIIIαdef(缺失了PI4KIIIα基因及其附近DNA的突变，Bloomington Stock#9518)、PI4KIIIαGS27和PI4KIIIαGJ86(均为无义突变)。P{lacW}l(2)k14710k15603转座子插在(l(2)k14710基因的第一个外显子中，阻止(l(2)k14710基因的转录，Bloomington Stock#11134)；P{EPgy2}bin3EY09582(Bloomington Stock#20043)。为了纯化基因背景，所有的转基因和突变体果蝇在使用前均分别与一个野生的等基因品系(isogenic w1118，Bloomington stock#5905)回交5代以上。
发明人之前的研究发现在GF通路中表达野生型或北极突变型(arctic mutant)Aβ42的果蝇(Aβ42或Aβarc果蝇)呈现神经元内Aβ42积累、日龄依赖性突触传递失败、寿命缩短。这些果蝇还表现出日龄依赖的爬行能力下降。为了研究rbo基因在神经元内Aβ42积累引起的神经障碍中的作用，将rbo基因的两个突变―错义突变(rbots1)和敲除突变(rbo2)―分别引入了Aβarc果蝇中，并分别检测了它们对突触 传递、爬行能力、寿命的影响。为了这些测试，共构建了4组果蝇，它们分别是对照果蝇(control)、rbots1/+或rbo2/+杂合体果蝇(rbo)、Aβarc果蝇和含有rbots1/+或rbo2/+杂合突变的Aβarc果蝇(Aβarc-rbo)。每组果蝇含有1-2个品系，其中，“ctrl”是指带有[Gal4]A307转基因的野生型对照组果蝇；“rbots1/+”and“rbo2/+”是指带有一个拷贝[Gal4]A307转基因的rbots1/+和rbo2/+杂合子果蝇；“Aβarc”是指带有([Gal4]A307/[UAS]Aβarc双转基因的果蝇；“Aβarc-rbots1/+”和“Aβarc-rbo2/+”是分别指[Gal4]A307-rbots1/[UAS]Aβarc和[Gal4]A307-rbo2/[UAS]Aβarc。前两组果蝇不表达Aβarc，被归类为“non-Aβflies”，而后两组果蝇是表达Aβarc的，被归类为“Aβflies”。
实施例2：通过突触传递检测rbo基因突变特异性地抑制Aβ42表达果蝇的神经障碍
突触传递的检测方法：
逃跑通路中记录细胞内的兴奋性突触后电位(excitatory junction potential,EJP)方法。利用低熔点蜡tackiwax(Boekel Scientific)将特定日龄的成年雌果蝇在解剖镜下固定在载玻片上，腹部向下。记录***包括腹部一根参考电极，***眼中的两根刺激电极和***背部纵向肌肉细胞内的一根记录电极。双眼给予电刺激(100Hz,50pulses)。刺激强度5-20Volts，持续时间0.2ms，约为阈值刺激强度的150％。电信号通过Axonal clamp 900A(Molecular Devices)记录并放大，在频率10kHz下通过Digidata 1440A(Molecular Devices)数字化。数据经由pClamp软件(version 10.0；Molecular Devices)记录并分析。电极均为玻璃电极，其中灌注3.0摩尔浓度的氯化钾溶液。记录的环境温度为25℃。
在图1中，4组果蝇在不同日龄阶段的脑刺激诱发的EJP的代表性记录(参见图1(a))，以及诱导EJPs的成功率的定量分析(参见图1(b))。特别值得注意的是，rbo突变明显抑制了由Aβarc引起的日龄依赖的神经突触传递失败。对于第3-7、15-17和25-27天的数据，n＝6～12，对 于31-35天的数据，n＝10-23，统计学检验使用的是单因素方差(one-way ANOVA)分析。
根据上述突触传递的检测方法，在果蝇大脑给予高频电刺激(100Hz，50个脉冲)，在背部纵向肌纤维细胞内记录兴奋性神经肌肉接头的终板电位(EJP)。当果蝇处于羽化后3-7th、15-17th、25-27th和31-35th日龄时我们进行了EJP记录。于3-7th和15-17th日龄时，高频电刺激在Aβarc-rbo果蝇中的EJP诱发成功率与在其它三组果蝇中的无显著差异(图1a和b)。于25-27th和31-35th日龄时，Aβarc-rbo果蝇中的EJP诱发成功率比对照果蝇和rbo果蝇的要低，但比Aβarc果蝇中的显著增高(图1a和b)。
这些结果显示rbo基因突变缓解了神经元内Aβarc积累引起的日龄依赖性突触传递失败。这种缓解作用不可能是基因背景差异所致，因为通过与一个野生的等基因系(isogenic w1118)回交5代以上，用于建立上述4组果蝇的转基因果蝇和rbo突变体果蝇的基因背景早已基本被纯化。由于完全敲除rbo基因导致胚胎致死，所以无法研究完全敲除rbo基因对Aβarc引起突触传递障碍的影响。
实施例3：通过爬管试验检测rbo基因突变特异性地抑制Aβ42表达果蝇的神经障碍
爬管试验：
爬管能力的研究是通过测量10只果蝇从直立的透明塑料管底部向上爬行7秒后的高度的平均值。为此，开发了一套重复性高的果蝇爬管能力测试装置。该装置包括：1)一个长方形的金属框架(宽32cm，高21cm)，框架中垂直固定10根透明塑料管(内径2.1cm，高19.0cm)；2)一台电动马达，用来驱动金属框架在垂直方向上移动；3)一台步进驱动器，用来把电动马达的工作节奏控制在每隔1分钟使金属框架 连续快速上下移动各4次，每次上下移动预定的高度；4)一台录像机，用来记录爬管过程；5)一套分析软件，用来分析录像中特定时刻果蝇的爬管位置。实验时，每根透明塑料管中放置10只特定基因型的果蝇，塑料管均匀分布并固定于金属盒中。金属盒可以沿着垂直固定在基座上的两根金属杆上下滑动。在爬管实验中，步进驱动器控制电机运转，电机将金属盒提升5.8cm然后释放，金属盒依重力下落到原位置。当金属盒停止运动时，果蝇由于惯性掉落到管底部。在金属盒3秒内被抬升下落4次后，所有果蝇都位于管底部。随后，果蝇沿管壁向上爬行。整个过程被录像以进行后续分析。发明人编写了一个电脑程序，用于测量每只果蝇在开始爬管后任何时间点的所在高度。
在图1(c)中，rbo基因突变改善了日龄依赖的表达Aβarc的果蝇的爬管能力，n＝3(3组果蝇，每组10只)，单因素方差(one-way ANOVA)分析。
在果蝇羽化后第3天、第16天、第26天和第31天测试果蝇的爬管能力。在第3天和第16天，四组果蝇的爬管能力在都相似(图1c)。在第26天和31天，Aβarc-rbo果蝇比Aβarc果蝇明显爬得快，但是并不像对照组和rbo果蝇那么快(图1c)。
实施例4：通过寿命分析rbo基因突变特异性地抑制Aβ42表达果蝇的神经障碍
寿命分析：
每种基因型100或200只果蝇，平均分配到5或10个含标准果蝇食物和干酵母的管中，在25℃下培养。每3天将果蝇换到含新食物和干酵母的管中，并记录死亡果蝇数目。最终用SPSS 11Kaplan-Meier软件统计存活率。
在图1(d)中，rbo基因突变延长了表达Aβarc的果蝇的寿命。每组 果蝇均是n＝200，P<0.001，采用Log Rank检验。
在图7中，3组果蝇在不同日龄阶段的脑刺激诱发的EJP的代表性记录(参见图7(a))，以及诱导EJP成功率的定量分析(参见图7(b))，“ctrl”是指带有[Gal4]A307转基因的野生型对照组果蝇；“Aβ42”是指带有([Gal4]A307和[UAS]Aβ42双转基因的果蝇；“Aβ42-rbots1/+”和“Aβ42-rbo2/+”是指[Gal4]A307-rbots1/[UAS]Aβ42和[Gal4]A307-rbo2/[UAS]Aβ42。值得注意的是，rbo突变明显抑制了由Aβ42引起的日龄依赖的神经突触传递失败。对于第3-5天的数据，n＝6～12，对于37-39天的数据，n＝10-12，单因素方差(one-way ANOVA)分析。
在图7(c)中，rbo基因突变改善了表达Aβ42的果蝇的日龄依赖的爬管能力，n＝3，单因素方差(one-way ANOVA)分析。
在图7(d)中，rbo基因突变延长了表达Aβ42的果蝇的寿命。每组果蝇均是n＝200，P<0.001，采用Log Rank检验。
寿命研究显示Aβarc-rbo果蝇的寿命也比Aβarc果蝇的长，虽然比对照组和rbo果蝇的短(图1d)；在比较四组果蝇的寿命平均值时也可以得出相同的结论(表1)。这些结果与上述研究突触传递时的发现一致。对rbo基因突变对表达野生型Aβ42的果蝇的突触传递、爬管能力和寿命长度的影响做出进一步的研究，发现了更好的改善(图7a－d)。
表1.果蝇寿命均值及其比较
Figure PCTCN2015078058-appb-000004
在图8(a)中，rbo基因突变对在果蝇巨纤维通路过表达果蝇Tau蛋白引起的运动障碍(左侧)和过早死亡的影响。在图8(b)中，shibire基因突变对在果蝇巨纤维通路过表达Aβarc引起的运动障碍(左侧)和过早死亡的影响。寿命分析实验中，每组果蝇均是n＝100，采用Log Rank检验。
rbo基因突变对抗Aβ42的毒性作用不能归因于一种对神经元内毒性蛋白积累的普遍效应，因为rbo基因突变不能改善过表达Tau蛋白的果蝇的寿命缩短(图8a)。rbo基因突变对抗Aβ42的毒性作用也不能归因于一种可能基于突触功能或者细胞内吞功能下调的普遍效应，因为向Aβarc果蝇中导入shibire基因突变(shibirets1)不能缓解Aβarc果蝇过早死亡的现象(图8b)。与rbots1基因突变相同，shibirets1基因突变也引起温度依赖的突触传递、散装的胞吞作用以及运动功能改变。
通过实施例1-4，结果显示rbo基因突变或不足可以特异性地改善表达野生型和突变体Aβ42果蝇的神经退行性病变。
实施例5：通过免疫共沉淀检测缺乏或抑制与RBO蛋白相互作用的PI4KIIIα酶减轻Aβarc果蝇的神经损伤
免疫共沉淀和免疫印迹：
收集300个果蝇头，在预冷的500μl Tris缓冲液中碾磨均匀。Tris缓冲液配方如下：50mM Tris,50mM KCl,1mM EDTA,1％cocktail protease inhibitor(Calbiochem)，调PH至7.4。将组织匀浆10000g离心10分钟，取上清，使用约1μg的小鼠源抗果蝇RBO单克隆抗体或者兔源抗果蝇PI4KIIIα的多克隆抗体进行免疫共沉淀和免疫印迹实验。上述两种抗体分别与上海Abmart和中国Abgent合作构建。构建RBO抗体采用的多肽是果蝇C亚型RBO蛋白的251th-500th氨基酸，构建抗果蝇PI4KIIIα的多肽是NH2-KRSNRSKRLQYQKDSYC-CONH2(SEQ ID  NO：3)。免疫印迹实验时，抗果蝇RBO和PI4KIIIα抗体的稀释比例都是1:2000。利用野生型和相应的纯合突变体的头部组织匀浆分别验证了抗果蝇RBO和PI4KIIIα抗体。
在图9(a)中，rboS358A基因不改善表达Aβarc果蝇的寿命(P＝0.07)。
尽管RBO是一种推定(putative)的二酯酰甘油(diacylglycerol,DAG)裂解酶，并且有报道说DAG裂解酶活性在AD病人和动物模型中的海马区有上调。但是，RBO蛋白不太可能以DAG裂解酶的作用方式调节Aβarc的毒性，因为在Aβarc果蝇中导入rboS358A转基因不能缓解Aβarc果蝇过早死亡的现象(图9a)。在rboS358A基因编码的RBO蛋白中，一个推定的酶活性中心被突变了。
在图1(e)中，显示RBO蛋白与PI4KIIIα蛋白相互共免疫沉淀的代表性免疫印迹图，n>3。
在图1(f)中，代表性免疫印迹图(左侧)和半定量分析(右侧)显示RBO蛋白与PI4KIIIα蛋白在野生型对照果蝇和rbo基因突变的杂合子果蝇中的表达量，n＝7，单因素方差分析。
在图1(g)中，代表性免疫印迹图(左侧)和半定量分析(右侧)显示PI4KIIIα分别与野生型(wtRBO)和突变型(mRBO)RBO蛋白的免疫共沉淀，n＝4，T检验。
在图1(h)中，RT-PCR定量分析PI4KIIIα信使RNA在Aβarc、Aβarc-rbots1/+、Aβarc-rbo2/+果蝇中的表达水平，n＝5-6，单因素方差分析。
RBO蛋白在酵母细胞和小鼠中的同源蛋白与PI4KIIIα蛋白结合，并且在细胞膜上形成一种复合体。与此一致的是，RBO蛋白会特异性地和果蝇PI4KIIIα蛋白发生免疫共沉淀(图1e)。此外，在Aβarc-rbo 果蝇中除去一个拷贝的rbo基因(rbo2/+)，可以显著地降低RBO蛋白和PI4KIIIα蛋白的表达水平(图1f)，而rbots1/+基因突变并没有减少RBO蛋白和PI4KIIIα蛋白的表达量，但是显著地削弱了RBO蛋白与PI4KIIIα蛋白之间的相互作用(图1g)。值得注意的是，两种rbo基因突变并没有改变PI4KIIIα基因的转录(图1h)。
为了测试PI4KIIIα酶在Aβarc果蝇的神经损伤中是否起着与RBO蛋白相同的作用，向表达Aβarc的果蝇中分别引入一种染色体缺失(一条染色体上缺失了包含PI4KIIIα基因的DNA片段，pi4kdef/+)和一种PI4KIIIα的无意义突变(PI4KIIIαGS27/+)。
在图2中，表达Aβarc的果蝇的突触传递和运动障碍及过早死亡均被杂合的PI4KIIIα基因缺失(PI4KIIIαdef/+)(参见图2(a))和无义(PI4KIIIαGS27/+)(参见图2(b))突变缓解，也被PAO缓解(图2(c)-(e))。“ctrl”是指带有[Gal4]A307转基因的野生型对照组果蝇；“PI4KIIIαdef/+”和“PI4KIIIαGS27/+”是指带有一个拷贝[Gal4]A307转基因的PI4KIIIαdef/+和PI4KIIIαGS27/+杂合子果蝇；“Aβarc”是指带有([Gal4]A307/[UAS]Aβarc双转基因的果蝇；“Aβarc-PI4KIIIαdef/+”和“Aβarc-PI4KIIIαGS27/+”是指PI4KIIIαdef/+；[Gal4]A307/[UAS]Aβarc和PI4KIIIαGS27/+；[Gal4]A307-rbo2/[UAS]Aβarc。对于每组EJP记录数据，n＝6～10；每个爬管实验数据，n＝3～5；每个果蝇品系的寿命数据，n＝100～200，P值均小于0.001。统计数检验方法如上所述。
事实上，实验结果表明这些PI4KIIIα的基因突变均抑制了Aβarc在突触传递、运动功能以及寿命上的引起的改变(图2(a)-(b))。与此结果一致的是，喂给Aβarc果蝇一种PI4KIIIα的抑制剂PAO，同样显著地减轻了这些改变，并呈现一种与药剂量相关的效应(图2(c)-(e))。
在图9(b)中，itprSV35基因突变不改善表达Aβarc果蝇的突触传递和寿命(P＝0.13)。寿命分析实验中，每组果蝇均是n＝100，采用Log Rank 检验；EJP诱发成功率实验中，n＝5。
然而，下调RBO/PI4KIIIα所带来的抑制Aβarc果蝇中的神经损伤不能够归结于减轻了磷脂酶C，PI4,5P和肌醇三磷酸受体(IP3R)所介导的钙释放所可能引起的毒性作用，因为向表达Aβarc的果蝇中引入编码肌醇三磷酸受体的基因的无意义突变即不能减轻突触障碍，也不能延长寿命(图9(b)-(c))。
实施例6：通过染色和成像检测下调RBO/PI4KIIIα减少了Aβ42的细胞内沉积
染色和成像：
果蝇中枢***中的Aβ染色方法如下。在预冷的PBS中解剖出果蝇中枢神经***(CNS)整体，包括脑和腹神经节，于PBS配置的4％PFA中固定约45分钟。以PBS洗30分钟，随后以甲酸(70％的水溶液)处理45分钟，使抗原决定簇暴露，然后用PBS-0.25％Triton-5％BSA重复冲洗。一抗(6E10,1:100稀释)在4℃下孵育10-12小时。以PBS洗，其后在室温下，以cy3-偶联的二抗(Jackson ImmunoResearch,1:200稀释)孵育2小时。使用Nikon A1R-A1共聚焦显微镜采集图像，果蝇中枢神经***的基因型对于采集图像的人员来说是未知的。
在图3(a)中，日龄为21-25天的表达mCD8-GFP的对照果蝇(上排)和Aβarc表达果蝇(中排)的腹侧神经节的全标本包埋Aβ染色的共聚焦成像；mCD8-GFP和Aβarc的表达均为[Gal4]A307驱动。每组染色重复两次；下排是中排图中被正方形框定区域的放大。日龄为21-25天的Aβarc、Aβarc-rbots1/+、Aβarc-rbo2/+、Aβarc-PI4KIIIαdef/+和Aβarc-PI4KIIIαGS27/+果蝇的代表性腹侧神经节的全标本包埋Aβ染色的共聚焦成像，每组染色重复两次(参见3(b))，以及ELISA方法定量头部的Aβ水平(图3(c)，上图)。图3(c)的下图是ELISA方法定量不同浓度PAO处理后的Aβarc表达果蝇头部的Aβ水平。ELISA方法定量 的每组数据，n＝3～5，单因素方差(one-way ANOVA)分析。在图3(a)和(b)中，比例尺是50微米。
先前，由于表达Aβarc在GF通路里所引发的神经损伤被归结于细胞内Aβ淀粉样蛋白的积累。这里，通过向Aβarc果蝇中导入uas-mCD8-gfp转基因，以便进一步证实Aβ淀粉样蛋白在神经元内积累。uas-mCD8-gfp转基因表达的是定位到细胞膜的mCD8-GFP萤光蛋白，它是由与Aβarc相同的驱动子驱动的，所以表达Aβarc的神经元会被GFP标记，共聚焦显微成像揭示绝大部分Aβ免疫染色信号与GFP信号共定位(图3(a))，从而确证了这一果蝇模型中神经元内Aβ积累的现象。
为了研究RBO/PI4KIIIα的不足是否会影响细胞内Aβ积累，在Aβarc，Aβarc-rbo,和Aβarc-PI4KIIIα果蝇中进行Aβ免疫染色。发现Aβarc-rbo和Aβarc-PI4KIIIα果蝇的Aβ免疫反应信号比Aβarc果蝇的明显减弱(图3(b))。
实施例7：通过ELISA定量测试检测下调RBO/PI4KIIIα减少了Aβ42的细胞内沉积
ELISA方法检测：
ELISA使用的是Aβ42Human ELISA Kit(invitrogen)，并按照使用说明进行实验。为分析中枢***中Aβ42的水平，将每个品系中25个果蝇的大脑在预冷过的PBS中解剖出来，并迅速置于冷的添加了蛋白酶抑制剂混合物(Calbiochem)的ELISA样品稀释液中，果蝇脑被充分的捣碎，室温摇动孵育4小时候，-20℃保存。
与实施例6相一致的是，ELISA定量测试显示在Aβarc-rbo,Aβarc-PI4KIIIα和用药物PAO治疗的Aβarc果蝇中，Aβ42的量显著地降低(图3(c&d))。
在图10(a)中，RT-PCR方法检测N2细胞中敲减EFR3a基因效率的代表图(左侧)和归一化法定量分析(中间)，右侧显示敲减EFR3a基因不影响N2a细胞内吞细胞外Aβ42。用于构建敲减Efra RNAi的序列是：5’-AGGTATCATTCAGGTTCTGTT-3’(SEQ ID NO:4)。图10(b)中，RT-PCR方法检测显示rbo基因及PI4KIIIa基因突变没有减低Aβarc表达果蝇中Aβarc转录水平。
通过实施例6和7，RBO/PI4KIIIα下调所导致的神经元内Aβ积累减少并不像是因为细胞外Aβ42的摄取的降低所导致，也不像是Aβarc转录的减少所造成，因为：1)敲减了rbo同源基因的N2a细胞摄取细胞外的Aβ42没有明显减少(图10(a))；2)在不同日龄的Aβarc-rbo以及Aβarc-PI4KIIIα果蝇中Aβarc的mRNA表达水平与实验组Aβarc果蝇比较并没有减少(图10(b))。
实施例8：PI4KIIIα抑制剂PAO、A1和G1溶液制备及其毒性测试
利用PAO或A1处理HEK293细胞、果蝇幼虫和成虫时，首先把PAO粉末(Sigma-Aldrich,CAS NO.637-03-6)和A1粉末(分别溶于DMSO，制成10和0.9mM的母液。然后用蒸馏水梯度稀释到所要的浓度，并调整DMSO的最终浓度以避免DMSO浓度差异影响实验结果。
测试PAO对活体果蝇的毒性时，我们用含有50、100、200、300、400和600μM PAO的食物培养野生型果蝇，从胚胎期开始。发现200μM及以下PAO对羽化率和羽化后的爬行能无明显影响。从而选取25、50、100和150μM PAO浓度培养Aβarc果蝇及对照果蝇。
测试PAO对解剖的果蝇三龄幼虫的毒性时，我们用含有50、100、150、200、300、400和500nM PAO的施耐德培养基在25℃下过夜培养解剖的野生型果蝇三龄幼虫。发现300nM和以上浓度的PAO会使果蝇幼虫的唾液腺和中枢神经***的颜色变为灰白，提示损伤。而150nM 和以下浓度的PAO则不会。因此，选取50、100和150nM PAO浓度进行培养。
测试PAO和A1对HEK239细胞的毒性时，用含有50、100、150、200、250、300、400和600nM PAO或A1的DMEM培养基培养HEK239细胞12小时后，用MTT法分析发现250nM和以上浓度的PAO和A1均会杀死大部分细胞，150nM和以下浓度则不会。因此，选取25、50、100和150nM PAO和A1浓度进行培养。
测试PAO灌胃对小鼠的毒性时，把PAO粉末溶于DMSO，制成30mg/ml的母液。然后用蒸馏水梯度稀释到所要的浓度，并调整DMSO的最终浓度以避免DMSO浓度差异影响实验结果。首先按18、10和6mg/kg体重的剂量给3月龄的C57BL/6小鼠灌胃，每个剂量灌胃2只小鼠。第2天发现所有小鼠均死亡。然后按4.5和2.0mg/kg体重的剂量灌胃，每个剂量灌胃5只小鼠。对于4.5mg/kg体重剂量，每天灌胃1次，连续5天后，5只小鼠中有4只存活。对于2.0mg/kg体重剂量，按周1至周5每天灌胃1次，周末停药。连续2周后，发现5只小鼠均存活。因此，对于C57/B6小鼠，PAO灌胃的半数致死量在2-6mg/kg体重，约为4mg/kg体重。所以，我们选取0.1、0.3和1.0mg/kg体重剂量给APP/PS1和对照小鼠灌胃，按周1至周5每天灌胃1次，周末停药，连续6周。
实施例9：通过表达Aβ的幼虫组织培养检测下调RBO/PI4KIIIα促进Aβ42分泌
表达Aβ的幼虫组织培养：
将果蝇三龄幼虫用水清洗和70％酒精消毒2分钟，在施耐德(Schneider’s)(Sigma)培养基中沿着幼虫的背中线剪开，小心清除幼虫的气管，内脏和脂肪组织。在施耐德培养基中清洗解剖过的幼虫，并转入2ml的离心管中，该离心管中有150ul的施耐德培养基和庆大霉 素(20毫克/毫升)。每管中有5个解剖后的幼虫，将离心管置于潮湿，黑暗，恒温25℃的环境中8小时。然后从每管中提取100ul用ELISA定量测试Aβ42。ELISA使用的是Aβ42Human ELISA Kit(invitrogen)。
在图4中，图4(a)-(c)显示归一化法定量分析不同PAO浓度处理、rbo基因和PI4KIIIα基因突变对培养解剖了的表达Aβarc果蝇三龄幼虫的培养基中Aβ42的水平的影响。
为了探究PAO药物治疗和RBO/PI4KIIIα不足减少神经元内Aβ积累的机理，检测了表达Aβarc的三龄幼虫解剖样本在Schneider培养基培养的过程中分泌Aβ42的情况。ELISA测试表明，PAO处理会促进Aβ42分泌，且呈现一种与药物剂量关联的趋势(图4(a))，表明抑制PI4KIIIα酶活性促进Aβ42分泌。与此一致的是，较之Aβarc幼虫的培养基，Aβarc-rbo品系和Aβarc-PI4KIIIα品系的幼虫培养基中有显著升高的Aβ42浓度(图4(b)-(c))。
实施例10：通过表达人源APP的HEK293T细胞培养检测下调RBO/PI4KIIIα促进Aβ42分泌
表达人源APP的HEK293T细胞培养：
稳转了人源APP的HEK293T细胞使用DMEM培养基(Hyclone)培养，加入10％FBS(Gibco)，青霉素和链霉素，和G418(100微克/毫升)。表达目的基因shRNA的pSUPER.basic重组质粒通过LipofectamineTM2000(invitrogen)瞬时转染到HEK293T细胞中。转染后培养细胞两天，再用于后续试验。用ELISA检测培养液中Aβ42浓度时，收集新换的且培养了细胞12小时的培养液。
APP的α,β,γ分泌酶的活性和稳转人源APP的HEK293T细胞中APP表达量检测：
利用12孔板培养稳转APP的HEK293T细胞，培养液中含有PAO 的浓度为0、25、50、100或150nM，培养6-8小时后，分别收集等量的细胞。检测分泌酶活性时，用500微升TBS缓冲液分别裂解细胞，低温离心(13000g)15分钟，保留上清，沉淀则用500微升TBS缓冲液重悬浮。检测α和β分泌酶的活性时，取100微升的上清与含有10μM的α或β分泌酶特异性萤光底物(specific fluorogenic substrates Calbiochem,Cat.No.565767/565758)的2倍反应液(50mM Tris-HCl,pH 6.8,4mM EDTA,0.5％CHAPSO(w/v))混合；检测γ分泌酶的活性时，取100微升的重悬浮液与含有10μM的γ分泌酶特异性萤光底物(specific fluorogenic substrates Calbiochem,Cat.No.565764)的2倍反应液(50mM Tris-HCl,pH 6.8,4mM EDTA,0.5％CHAPSO(w/v))混合。在37℃反应30分钟后，用酶标仪(excitation/emission：365/490nm forα/β酶活性，365/440forγ酶活性)检测。
检测APP表达量时，将收集的细胞用含有蛋白酶抑制剂(1％cocktail，invitrogen)TBS缓冲液裂解后进行后利用抗APP/Aβ抗体(6E10)进行免疫印迹。
图14(a)示归一化法定量分析PAO对HEK293T细胞的α,β,γ分泌酶活性的影响，每个数据点，n＝5，单因素方差检验；图14(b)示免疫印迹显示经不同PAO浓度处理后HEK293T细胞的APP表达量的代表图，该实验重复了3以上。
为了测试这种易化作用是否作用于从β淀粉样蛋白前体蛋白(APP)剪切而来的Aβ42的分泌，检测了一种稳定过量表达人源APP的HEK293T细胞的Aβ42分泌情况。在图4(d)-(g)中，归一化法定量分析不同PAO和A1浓度处理、EFR3a和PI4KA基因敲减对培养稳转人源APP的HEK293细胞的培养基中Aβ42的水平的影响。事实上，PAO处理在培养基中产生类似的提高Aβ42浓度的效果(图4(d))，且PAO即使在γ-分泌酶抑制剂DAPT(1uM)存在的情况之下，依然提升了培养基中的Aβ42浓度(图4(e))。值得注意的是，PAO的浓度甚至在相当低(甚至可以低到1.0nM)的情况下，仍然存在稳定的效果(图4(i))。再者，在稳定过量表达人源APP的HEK293T细胞中敲减EFR3a或 PI4KA基因或应用A1处理均可以显著的提升培养基内的Aβ42浓度(图4(f-g)。每组数据，n＝4～15，单因素方差检验。
根据检测在稳定过量表达人源APP的HEK293T细胞的Aβ42分泌情况，对以下化合物进行检测并获得如下结果：
Figure PCTCN2015078058-appb-000005
Figure PCTCN2015078058-appb-000006
Figure PCTCN2015078058-appb-000008
Figure PCTCN2015078058-appb-000009
Figure PCTCN2015078058-appb-000010
Figure PCTCN2015078058-appb-000011
Figure PCTCN2015078058-appb-000012
Figure PCTCN2015078058-appb-000013
Figure PCTCN2015078058-appb-000014
此外，PAO促进稳转APP的HEK293细胞分泌Aβ42，但不改变剪切APP的α,β,γ分泌酶的活性图14(a)、也不引起APP水平升高图4(b)。
实施例11：病毒构建包装及其在小鼠中的显微注射
慢病毒由invitrogen公司(上海)使用BLOCK-iTTM HiPerformTM Lentiviral Pol II miR RNAi Expression System with EmGFP这一表达***制造。四个针对EFR3a基因的miRNAs寡聚片段被合成并***pcDNATM6.2-GW/EmGFPmi载体。用RT-PCR或Western Blot的方法检测敲除效率。检测结果显示，其中一个载体在过表达EFR3a基因的HEK293T细胞中，对沉默EFR3a基因表达最有效。敲除效率最高的载体目的序列为AGGTATCATTCAGGTTCTGTT。这一最有效的miRNA载体与pDONRTM221及pLenti6/V5DEST重组，通过
Figure PCTCN2015078058-appb-000015
重组反应得到pLENT6/V5-GW/±EmGFP-miRNA载体。慢病毒通过共转染pLENT6/V5-GW/±EmGFP-miRNA vector和Packaging Mix得到。病毒浓度经由在HEK293T细胞中连续稀释得到。随后每三天统计一次EGFP阳性的细胞。沉默效率进一步通过慢病毒转染原代培养的海马神经元得到。
APP/PS1转基因雄鼠(B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax(MMRRC ID 034832-JAX)通过与(C57BL/6和C3H交配的F1带)小鼠杂交保种。在6月龄时，使用100mg/kg Ketamine加20mg/kg  Xylazine麻醉小鼠，固定于立体定位仪，腹部向下置于电热毯上。清除头部毛发，切开皮肤，穿透颅骨打一小洞。使用注射泵(Harvard Apparatus)通过套管***(external diameter,0.29mm,internal diameter,0.1mm,RWD Life Science Co.,Ltd)，在20分钟内注射2μl慢病毒溶液(病毒浓度：6x10-7)至2.1mm posterior to bregma,2.3mm lateral and1.9mm ventral。注射后5分钟，移除针头，缝合皮肤，小鼠移至25℃含充足食物和水的环境中培养。至12月龄时，再次麻醉小鼠，使用PBS配制的4％多聚甲醛(para-formaldehyde,简称PFA)灌流。实验遵照美国神经科学协会对动物使用的政策在小鼠上进行实验。
实施例12：通过小鼠脑片GFP染色检测敲减EFR3a基因可修复APP/PS1鼠神经元树突的萎缩
小鼠脑片GFP染色：
脑片(厚60μm)使用PBS-0.3％triton-5％BSA封闭1小时，使用兔源anti-GFP抗体(A11122,invitrogen,1:100稀释)于4℃孵育过夜。随后以PBS洗，并用生物素化羊源抗兔IgG抗体(H+L)(AbboMax,Inc,1:100稀释)于4℃孵育过夜。再次以PBS洗，使用Cy3-Streptavidin(Jackson ImmunoResearch Laboratories Inc,1:1000稀释)于室温下孵育2小时。使用Zeiss LSM 511共聚焦显微镜采集图像，通过AutoQuant X2反卷积，再用NeuronStudio软件进行数据分析。脑片与采集到的树突图像所属基因型对于采集图像的人员和进行数据分析的人来说都分别是未知的。
在图11中，RT-PCR方法检测在HEK293细胞中敲减内在EFR3a基因(a)和PI4KA基因(b)，在HEK293细胞中敲减过表达的小鼠EFR3a-gfp重组基因(c)，以及在小鼠原代培养的海马神经元中敲减小鼠内在EFR3a基因(d)的效率。代表图在上，归一化法定量分析在下。用于构建敲减人源Efr3a和PI4KA基因的RNAi的序列分别是：5’-GGTTATTGAAATTCGAACT-3’(SEQ ID NO:5)和5’-TGCTCATT AGCAGTAAAGA-3’(SEQ ID NO:6)。对于每个数据，n＝3-5，T检验获得P值。
在图5(a)中，共聚焦成像显示抗GFP免疫染色的海马切片全图(上部)与慢病毒转染的CA3区锥体细胞(下部)。选取长度约30μm的树突片段(两箭头之间)片段量化树突的直径和树突棘的密度。比例尺是500微米(上部)和50微米(下部)。在APP/PS1和同窝对照小鼠的CA3区锥体神经元(参见图5(b))和DG区颗粒神经元(参见图5(c))中敲减EFR3a基因对树突直径和树突棘密度的影响。CA3和DG区树突的代表图位于顶部，树突直径和树突棘密度的定量分析位于底部。每个数据点取自3～4只动物的n≥25张切片，P值通过单因素方差分析得到。比例尺在图5(b)和(c)中是1.0微米。
小鼠和人类有EFR3a基因和EFR3b基因两个rbo的同源基因，这两个基因在海马等AD易感区富集(Allen brain atlas)。通过在APP/PS1小鼠中运用EGFP标记的RNAi技术，研究下调EFR3a基因后能否保护海马神经细胞。RNAi的敲减效率(见图11(a)&(b))。激光共聚焦成像显示海马CA3和DG区的小部分神经细胞被表达EGFP的慢病毒感染(图5(a)上)。我们通过随机选取细胞近端部分(约30μm长)的次级顶树突，测量其树突直径及树突棘密度。在野生型对照组小鼠与APP/PS1小鼠之间比较，以上两个参数的值在受对照慢病毒感染的APP/PS1小鼠的CA3和DG区神经细胞中显著降低(图5(b)&(c))，表明APP/PS1小鼠的海马神经元树突及树突棘有萎缩。在APP/PS1小鼠之间比较，以上两个参数的值在受RNAi慢病毒感染的神经细胞中显著增高，几乎与这两个参数在野生型小鼠中的值无差异(图5(b)&(c))。因此，下调APP/PS1小鼠海马的神经元的rbo同源基因EFR3a也可保护神经细胞。
实施例13：通过对脑脊液和分离小鼠大脑细胞膜组分的分析检测PAO 改善APP/PS1小鼠的记忆和学习能力，增加脑脊液内Aβ42，但减少大脑细胞膜中的Aβ42
脑脊液的采集：
用***和甲苯噻嗪将小鼠麻醉，注意用适配器保护小鼠的头部。将小鼠颈部的毛剃掉并切开皮肤，用钳子把下面的皮下组织和肌肉向两侧分开，以暴露覆盖在小脑延髓池的那部分硬脑膜。准备一只一端很锐利但另一端较钝的玻璃毛细管，钝的一端连接显微注射器，然后用锐利的一端刺入硬脑膜。当感觉到明显的突破感时，说明毛细管成功进入小脑延髓池，脑脊液就会被吸入毛细管，大约收集10－20微升。将收集的脑脊液转移到微量离心管，-80℃保存备用。
分离小鼠大脑细胞膜组分：
通过一系列的抽提得到可溶于去垢剂的Aβ42，加入相当于小鼠大脑半球的5倍体积的用缓血酸胺缓冲液(TBS)进行研磨，研磨成匀浆后放入离心机，在4℃以100,000g离心60分钟，则上层清液是TBS提取物。收集下面的沉淀，加入5倍体积的含有1％聚乙二醇辛基苯基醚的TBS缓冲液，再次研磨并离心，则上层为TBS-Triton提取物。再收集下层沉淀，加入5倍体积的包含1％SDS的TBS缓冲液，进行第三次研磨并离心，则上层清液为TBS-SDS提取物。分别收集这三次的上次清液，分装好后，放入-80℃冰箱保存，为做ELISA检测备用。
以上在培养细胞和果蝇中的实验结果显示PAO可以促进Aβ42分泌，减少神经元内Aβ42积累并改善突触等功能，说明通过促进Aβ42分泌，PAO可作为治疗AD的一个潜在药物。为此，对灌胃服用了不同PAO剂量的APP/PS1小鼠进行了行为和生化实验。
在图6(a)中，不同浓度PAO处理的APP/PS1小鼠(左侧图)和同窝出生的野生型小鼠(右侧图)的水迷宫实验训练曲线。为了方便对比，PAO灌胃浓度为0的APP/PS1小鼠的学习曲线在左右两图中均有 显示。在图6(b)中，训练后对照和APP/PS1小鼠在目标象限中的搜寻时间占总的搜寻时间的百分比。ELISA方法定量分析不同浓度处理的APP/PS1小鼠脑脊液中(参见图(c))，和1％Triton及1％SDS的TBS缓冲液提取的大脑细胞膜组分中(参见图6(d))的Aβ42水平。在图6(e)中，100℃处理不同时间对ELISA方法定量分析APP/PS1小鼠脑脊液中(左图)和1％SDS的TBS缓冲液提取的大脑细胞膜组分中的Aβ42测定值的影响。在图6(f)中，ELISA方法定量分析100℃处理60分钟后1％Triton及1％SDS的TBS缓冲液提取的大脑细胞膜组分中的Aβ42水平。在图6(f)中，100℃处理60分钟后从1％Triton及1％SDS的TBS缓冲液提取的各大脑细胞膜组分中所释放出的Aβ42量占各自总量的百分比。在图6(a)和(b)中，各野生型对照组n＝6～8，各APP/PS1组n＝7～8；在图6(e)，左侧，n＝3，图e，右侧，n＝23；在图6(c,d和f)中，n＝5～6。P值均通过单因素方差(one-way ANOVA)分析得到。
通过在实施例8中描述的对野生型小鼠灌胃测试PAO的毒性后，以0、0.1、0.3和1.0mg/kg为浓度梯度进行以下实验。对4月龄大的APP/PS1小鼠和野生型小鼠进行PAO灌胃，周一至周五每天一次，连续6周。之后停药一周，通过按照Vorhees and Williams描述的方法进行的水迷宫实验测试小鼠的学***，尤其当剂量在0.3mg/kg时(图6(d))。但是，图6(f)显示PAO处理显著减少了大脑细胞膜中聚合状Aβ42的水平，尤其当剂量在0.3mg/kg时。
实施例14：通过水迷宫实验检测突变小鼠PI4KIIIα改善APP/PS1小鼠的学习记忆能力
在Aβ42表达果蝇中遗传方法下调PI4KIIIα的表达量或PAO抑制其酶活性都减轻了神经功能异常，在APP/PS1小鼠中PAO抑制PI4KIIIα的酶活性也改善了学习记忆功能。为了进一步明确PI4KIIIa在APP/PS1小鼠神经退行性改变中的功能，我们检测了PI4KIIIα杂合突变(Pi4kaGt(RRO073)Byg/+:转座子pGT2Lxf***在一个拷贝的PI4KA基因中会导致该拷贝基因在转录时出现障碍，产生的mRNA仅能翻译PI4KIIIα氨基端的前～265个氨基酸与报告基因编码的蛋白所组成的融合蛋白)对APP/PS1小鼠在水迷宫实验中影响。把Pi4kaGt(RRO073)Byg/+突变杂合子(MMRRC，Cat.#016351-UCD)与APP/PS1小鼠交配，获得四组基因型小鼠：野生型(WT)、PI4KIIIa突变杂合子(PI4K*/+)、APP/PS1(TG)和含有PI4KIIIa杂合突变的APP/PS1(TG；PI4K*/+)。当这四组小鼠达到5月龄时按照Vorhees and Williams描述的方法进行水迷宫实验。如图12所示，结果显示PI4KIIIa杂合突变能显著改善5月龄APP/PS1小鼠的空间学习和记忆能力。图12左侧显示的是四组基因型小鼠在训练日的上台时间，右侧显示的是在训练结束日后第1天的测试中四组基因型小鼠在目标象限中游动时间所占的百分比。
实施例15：通过脂质体实验检测PI、PI4P、PI4,5P在脂质体中对Aβ42聚合的影响
脂质体实验：
1)用六氟异(2)丙醇(hexafluoro-2-propranol，HFIP)溶解合成的Aβ42，溶解比率是1:1(mg:ml)，然后是在室温下用50Hz的超声波混匀15分钟。超声波产生器是KUDOS ultrasonic instrument(Model SK250HP)。
2)用氯仿(chloroform)分别溶解用于制备脂质体的各种脂类(见 表1)溶解比率也是是1:1(mg:ml)，在室温下用50Hz的超声波混匀15分钟。
3)按表1的比率把Aβ42和各种脂类混合在一起，随后用冷冻干燥器抽干混合液中的有机溶剂。
4)用1.0ml的Tris缓冲液(50mMTris,120mM NaCl,pH7.0)重悬浮冷冻干燥处理后所得的沉淀，在冰水中超声波混匀30分钟。然后在4℃下静置48小时后进行免疫印迹检测(一抗是抗人源Aβ的单可容抗体6E10，1:1000)。免疫印迹检测前需在冰水中超声波混匀15分钟。
表1.制备脂质体的各种脂类配比
Figure PCTCN2015078058-appb-000016
对PI、PI4P、PI4,5P在脂质体中对Aβ42寡聚体形成的影响和比较进行分析，图13的左列显示的是PI4P对脂质体中Aβ42寡聚体形成的促进作用呈浓度依赖性，上下部是同一免疫印记膜的相对短和长时间的曝光后显影的结果；请注意PI4P浓度为80μM时，对Aβ42寡聚体形成的促进作用反而比40μM时有所减弱。图13的右列显示的是PI、PI4P和PI4,5P对脂质体中Aβ42聚合的促进作用，上下部是同一免疫印记膜的相对短和长时间的曝光后显影的结果；请注意PI4P的作用明显比PI和PI4,5P，且PI和PI4P对Aβ42三聚体和以上的寡聚体形成的促进作用 又比PI4,5P强。
实施例16：RBO/EFR3/EFR3A/EFR3B、PI4KIIIa和TTC7在细胞膜上形成复合体
报道显示，酵母的EFR3蛋白与PI4KIIIa和一个脚手架蛋白YPP1(在哺乳动物中叫TTC7，包括TTC7A和TTC7B两个同源蛋白)在细胞膜上形成复合体，且以斑片状聚集(PIK patchs)，一起控制膜的PI4P水平、甚至PI4,5P水平。YPP1与酵母PI4KIIIa蛋白的N端和中间区域均有直接相互作用，对PIK patchs的组建和稳定起着关键作用(Baird D,Stefan C,et al.,2008,J Cell Biol)。PIK patchs的构成和功能在哺乳动物细胞中也保守(Nakatsu F,Baskin JF,et al.,2012,J Cell Biol)。果蝇中的TTC7的果蝇同源蛋白是由lethal(2)k14710[(l(2)k14710)基因编码。
为了检测TTC7蛋白在神经元内Aβ积累引起的神经退行性改变中的作用，在Aβarc果蝇中分别引入两个转座子(transpon)介导的转基因，一个是P{lacW}l(2)k14710k15603,(Bloomington Cat.#11134)该转座子插在(l(2)k14710基因的第一个外显子中，阻止(l(2)k14710基因的转录；另一个是P{EPgy2}bin3EY09582,(Bloomington Cat.#20043)。该实验共构建了4组果蝇：对照果蝇(ctrl)、Aβarc果蝇、含有一个拷贝的P{lacW}l(2)k14710k15603的Aβarc果蝇(Aβarc-dttc7+/-，TTC7下调)和含有一个拷贝P{EPgy2}bin3EY09582的Aβarc果蝇(Aβarc-dttc7-OE，TTC7过表达)。当4组果蝇的成虫达到5-10、30-35天两个日龄段后，进行100赫兹脑刺激诱发的逃跑通路的兴奋性突触后电位记录。
图15示定量分析分别下调和过量表达TTC7对Aβarc果蝇中神经传递的影响。在日龄为5-10天时，4组果蝇的100赫兹脑刺激诱发的神经传递成功率无明显差异，但在日龄为30-35天时，Aβarc果蝇的神经传递的成功率明显比对照果蝇(ctrl)要低，过量表达TTC7 (Aβarc-dttc7-OE)使成功率进一步降低，但下调TTC7表达(Aβarc-dttc7+/-)则提高神经传递的成功率。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (14)

  1. 一种利用PI4KIIIα抑制剂治疗阿尔茨海默病的方法。
  2. 如权利要求1所述的方法，所述PI4KIIIα抑制剂为抗PI4KIIIα抗体、对PI4KIIIα特异的抑制性核苷酸、或对PI4KIIIα特异的小分子化合物抑制剂。
  3. 如权利要求2所述的方法，所述对PI4KIIIα特异的抑制性核苷酸为如SEQ ID NO：6所示的核苷酸序列。
  4. 如权利要求2所述的方法，所述PI4KIIIα抑制剂选自下列小分子化合物抑制剂中的一种或多种：PAO、PAO的衍生物、G1、A1、G1或A1的类似物。
  5. 一种利用RBO/EFR3/EFR3A/EFR3B抑制剂治疗阿尔茨海默病的方法。
  6. 如权利要求5所述的方法，所述RBO/EFR3/EFR3A/EFR3B抑制剂为抗RBO/EFR3/EFR3A/EFR3B抗体。
  7. 一种利用PI4P抑制剂治疗阿尔茨海默病的方法。
  8. 如权利要求7所述的方法，所述PI4P抑制剂为抗PI4P抗体、OSH2-PH2X融合蛋白、或者OSH2-2x-PH融合蛋白。
  9. 一种药物组合物，其包含下列物质的一种或多种：PI4KIIIα抑制剂、RBO/EFR3/EFR3A/EFR3B抑制剂以及PI4P抑制剂，还包含任选的药物载体。
  10. 如权利要求9所述的药物组合物，其还包含一种或多种抗Aβ抗体和/或能清除神经细胞外Aβ聚合物的化合物。
  11. 如权利要求10所述的药物组合物，所述能清除神经细胞外Aβ聚合物的化合物选自：海洋硫酸寡糖类HSH971及其类似物、阿坎酸及其类似物、以及依达拉奉及其类似物。
  12. 一种以PI4KIIIα蛋白的激酶活性为靶点的筛选阿尔茨海默病药物的方法，所述方法包括以下步骤：观察候选药物对PI4KIIIα蛋白的磷酸激酶活性的影响，若候选药物能够抑制PI4KIIIα的磷酸激酶活性，则表明该候选药物为治疗阿尔茨海默病的潜在药物。
  13. 一种以RBO/EFR3/EFR3A/EFR3B蛋白和PI4KIIIα蛋白的相互作用为靶点的筛选阿尔茨海默病药物的方法，包括以下步骤：观察候选药物对RBO/EFR3/EFR3A/EFR3B蛋白和PI4KIIIα蛋白相互作用的影响，若候选药物能够抑制RBO/EFR3/EFR3A/EFR3B蛋白和PI4KIIIα蛋白的相互作用从而减少RBO/EFR3/EFR3A/EFR3B-PI4KIIIα复合蛋白的形成，则表明该候选药物是治疗阿尔茨海默病的潜在药物。
  14. 一种以细胞膜上的PI4P水平为靶点的筛选阿尔茨海默病药物的方法，所述方法包括以下步骤：观察候选药物是否对细胞膜上的PI4P水平有影响，若候选药物能够抑制细胞膜上的PI4P水平，则表明该候选药物是治疗阿尔茨海默病的潜在药物。
PCT/CN2015/078058 2015-04-30 2015-04-30 PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用 WO2016172952A1 (zh)

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US15/570,681 US11766420B2 (en) 2015-04-30 2016-05-03 Application of PI4KIIIA protein and related membrane protein complex in treating alzheimer's disease
RU2017141805A RU2724493C2 (ru) 2015-04-30 2016-05-03 Изобретение белка pi4kiiia и родственного мембранного белкового комплекса в лечении болезни альцгеймера
JP2018507766A JP6755938B2 (ja) 2015-04-30 2016-05-03 アルツハイマー病の治療におけるＰＩ４ＫＩＩＩαタンパク質および関連膜タンパク質複合体の利用
KR1020177034632A KR102048137B1 (ko) 2015-04-30 2016-05-03 알츠하이머 질환 치료시 PI4KIIIα 단백질 및 관련 막 단백질 컴플렉스의 적용
AU2020202847A AU2020202847B2 (en) 2015-04-30 2020-04-30 Application of PI4IIIA protein and related membrane protein complex in treating Alzheimer's disease
JP2020142213A JP2020203907A (ja) 2015-04-30 2020-08-26 アルツハイマー病の治療におけるＰＩ４ＫＩＩＩαタンパク質および関連膜タンパク質複合体の利用
JP2022161004A JP2023011586A (ja) 2015-04-30 2022-10-05 アルツハイマー病の治療におけるＰＩ４ＫＩＩＩαタンパク質および関連膜タンパク質複合体の利用

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