WO2006053063A2 - Dosage de notch-1 utilise dans la detection de maladies neurodegeneratives - Google Patents

Dosage de notch-1 utilise dans la detection de maladies neurodegeneratives Download PDF

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WO2006053063A2
WO2006053063A2 PCT/US2005/040625 US2005040625W WO2006053063A2 WO 2006053063 A2 WO2006053063 A2 WO 2006053063A2 US 2005040625 W US2005040625 W US 2005040625W WO 2006053063 A2 WO2006053063 A2 WO 2006053063A2
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notch
nicd
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disease
prp
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WO2006053063A3 (fr
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Stanley B. Prusiner
Stephen J. Dearmond
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The Regents Of The University Of California
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • This invention relates generally to the field of neurodegenerative diseases and more particularly to assaying Notch- 1 to detect neurodegenerative diseases such as CJD in humans.
  • Prions are infectious proteins that propagate by recruiting a naturally occurring precursor protein and stimulating its conversion into nascent prions (Prusiner, S. B., Scott, M. R., DeArmond, S. J. & Cohen, F. E. (1998) Cell 93, 337-348). Formation of prions involves a conformational change in the precursor protein (Cohen, F. E., Pan, K.-M., Huang, Z., Baldwin, M., Fletterick, R. J. & Prusiner, S. B. (1994) Science 264, 530-531). In mammals, the accumulation of prions is accompanied by neurodegeneration (DeArmond, S. J., Mobley, W. C, DeMott, D.
  • the prion diseases include Creutzfeldt- Jakob disease (CJD) of humans, scrapie of sheep, and bovine spongiform encephalopathy (BSE).
  • CJD Creutzfeldt- Jakob disease
  • BSE bovine spongiform encephalopathy
  • PrP Sc the sole component of the prion, PrP Sc , accumulates in the CNS, resulting in presynaptic bouton degeneration, dendritic atrophy, vacuolation of neurons, and hypertrophy of astrocytes (DeArmond, S. J., Mobley, W. C, DeMott, D. L., Barry, R. A., Beckstead, J. H. & Prusiner, S. B.
  • PrP Sc is formed from the precursor protein PrP c by a profound conformational change (Cohen, F. E., Pan, K.-M., Huang, Z., Baldwin, M., Fletterick, R. J. & Prusiner, S. B. (1994) Science 264, 530-531). PrP c contains three ⁇ -helices (Donne, D. G., Viles, J. H., Groth, D., Mehlhorn, L, James, T. L., Cohen, F. E., Prusiner, S. B., Wright, P. E. & Dyson, H. J. (1997) Proc. Natl. Acad. Sci.
  • PrP Sc The tertiary structure of PrP Sc appears to encipher biological information that defines a particular prion strain (Legname, G., Baskakov, I. V., Nguyen, H. O., Riesner, D., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. (2004) Science 305, 673-6; Bessen, R. A. & Marsh, R. F. (1994) J. Virol. 68, 7859-7868; Telling, G. C, Parchi, P., DeArmond, S. J., Cortelli, P., Montagna, P., Gabizon, R., Lugaresi, E., Gambetti, P.
  • prion diseases are invariably fatal and other neurodegenerative diseases such as Alzheimer's disease are also fatal and widespread in the population, there remains very little technology with respect to the ability to accurately diagnose the presence of these diseases in a human patient. Often, the disease is confirmed by the examination of brain tissue after the patient or animal has died.
  • An assay is disclosed whereby Notch- 1 levels in a sample are determined and increases in Notch- 1 levels beyond normal levels are associated with an increased probability of a neurodegenerative disease being present.
  • the expression of Notch- 1 is blocked and/or its activity is suppressed thereby treating a patient suffering from neurodegenerative disease.
  • the detecting may be detecting the presence of Notch- 1 or an intercellular domain of Notch- 1 (NICD) and relating that detection to a positive result or disease state based on comparison to a normal control.
  • the method may be carried out by combining an antibody with binds to NICD of Notch- 1 with a sample obtained from a subject and allowing the antibody to remain with the sample for a period of time and under conditions so as to allow binding of the antibody to NICD present in the sample.
  • the binding is undetected which may be carried out in a number of different ways including labeling the antibody or using an labeled antibody which binds to the antibody binding to the NICD and thereafter detecting the label.
  • the detected binding in the sample is compared to a known standard to show a level of detection above a normal control which can provide a positive result or a diagnosis of neurodegenerative disease in the subject.
  • the detection can be visual as by detecting levels of NICD and its presence in different parts of a cell as compared to a normal cell.
  • an aspect of the invention is that the assay can be carried out by using a blood sample from the patient and as such can be carried out while the patient is still alive and may be experiencing relatively early symptoms of the disease.
  • the subject can be any animal including any mammal which may be a human, cow, sheep, goat, mule, deer, elk or other ungulate.
  • Another aspect of the invention is that it can be carried out relatively quickly, inexpensively and with the use of a relatively low degree of expertise.
  • results obtained with the assay can be visualized and thereby quickly analyzed to determine if the results are positive (neurodegenerative disease present) or negative indicating the lack of disease.
  • An aspect of the invention is an assay for detecting levels of Notch- 1 in a sample.
  • Another aspect of the invention is a method of treating a neurodegenerative disease by suppressing expression of Notch- 1.
  • Yet another aspect of the invention is a method of treating a neurodegenerative disease by inhibiting Notch- 1 activity.
  • Still another aspect of the invention is a formulation comprised of a carrier such as a pharmaceutically acceptable carrier and an inhibitor of Notch- 1 activity such as an antibody which binds to Notch- 1.
  • Figure 1 includes images IA of Western Blots, IB a bar graph and 1C confocal microscopy images of NICD accumulation.
  • Figure 2 includes graphs 2A, 2B, 2C, 2F and 2G and drawing 2D and 2E of Galgi- silver stained dendritic trees.
  • Figure 3 shows two Wester blots 3A, and four phase contrast micrscopy images 3B,
  • Figure 4 includes four Western blot images as 4A, two phase contrast microscopy images 4B and 4C; and a graph 4D.
  • Figure 5 shows two images of immunohistochemical staining for NICD in lymphocytes with the left photo showing the control and the right photo showing results from a human CJD patient.
  • Prions are defined as proteinaceous infectious particles that are devoid of nucleic acid and seem to be composed exclusively of a modified isoform of the prion protein designated p r p Sc Jj 16 J101 - J113 I 5 ⁇ eiiuiaj p r p denoted PrP c is converted into PrP Sc through a process whereby some of its ⁇ -helical structure is converted into ⁇ -sheet. This structural transition is accompanied by profound changes in the physico-chemical properties of the PrP. Whereas PrP c is soluble in nondenaturing detergents, PrP Sc is not. PrP c is readily digested by proteases whereas PrP Sc is partially resistant. The species of a particular prion is determined by the sequence of chromosomal PrP gene of the mammal in which is last replicated. Diseases caused by prions are listed in Table 1 below. Table 1 The prion diseases
  • HGH dura mater grafts, etc.
  • BSE bovine spongiform encephalopathy
  • CJD Creutzfeldt- Jakob disease
  • sCJD sporadic CJD
  • fCJD familial CJD
  • iCJD iatrogenic CJD
  • vCJD new) variant CJD
  • CWD chronic wasting disease
  • FSE fatal familial insomnia
  • FSE feline spongiform encephalopathy
  • FSI fatal sporadic insomnia
  • GSS Gerstmann-Straussler-Scheinker disease
  • HGH human growth hormone
  • MM meat and bone meal
  • TAE transmissible mink encephalopathy.
  • PrP Human PrP is encoded by a gene on the short arm of chromosome 20, and all other mammals examined also have a single-copy gene encoding PrP.
  • prions encipher strain-specific properties in the tertiary structure of p r p Sc ⁇ ranS g ene tic studies argue that PrP Sc acts as a template upon which PrP is refolded into a nascent PrP Sc molecule and that this process is facilitated by another protein or complex of proteins.
  • PrP Sc the conformation of PrP c is modified by PrP Sc as it is refolded into a nascent molecule of PrP Sc .
  • prions are infectious because they stimulate a process by which more of the pathogen is produced. As prions or viruses accumulate in an infected host, they eventually cause disease. Both prions and viruses exist in different varieties or subtypes that are called strains, but many features of prion structure and replication distinguish them from viruses and all other known infectious pathogens.
  • Prions differ from viruses and viroids because they lack a nucleic acid-genome that directs the synthesis of their progeny. Prions are composed of an abnormal isoform of a cellular protein, whereas most viral proteins are encoded by a viral genome, and viroids are devoid of protein.
  • Prions can exist in multiple molecular forms, whereas viruses exist in a single form with a distinct ultrastructural morphology. Prions have no constant structure, in marked contrast to viruses. Prion infectivity has been detected in fractions containing particles with an extremely wide range of sizes.
  • Prions are nonimmunogenic, in contrast to viruses that almost always provoke an immune response. Prions do not elicit an immune response because the host has been rendered tolerant to PrP Sc by PrP c . In contrast, the foreign proteins of viruses that are encoded by the viral genome often elicit a profound immune response. Thus, it seems unlikely that a useful strategy for preventing or treating prion disease will be vaccination, which has been so effective in preventing many viral illnesses.
  • excipient material and “carrier” are used interchangeably here and intended to mean any compound forming a part of the formulation which is intended to act merely as a carrier i.e. not intended to have biological activity itself.
  • treating and “treatment” and the like are used interchangeably herein to generally mean obtaining a desired pharmacological and physiological effect.
  • the effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e.
  • the invention is directed towards treating patient's suffering from a neurodegenerative disease and related to the effects of such a disease these over long periods of time.
  • the present invention is involved in preventing, inhibiting, or relieving adverse effects attributed to a neurodegenerative disease over long periods of time.
  • the terms "synergistic”, “synergistic effect” and the like are used interchangeably herein to describe improved treatment effects obtained by combining two or more compounds in formulations of the invention or two or more methods of treatment.
  • a synergistic effect in some fields means an effect which is more than additive (e.g., one plus one equals three) in the field of treating humans with a degenerative disease
  • an additive (one plus one equals two) or less than additive (one plus one equals 1.2) effect may be synergistic. If additive effects could always be obtained then neorodegenerative diseases could be readily treated in all instances by coadministering several different types of compounds. However, such has not been found to be an effective treatment.
  • coadministration of formulations of the invention to inhibit the activity of Notch- 1 and to suppress it expression provide improved the effects which are synergistic, i.e. greater than the effects obtained by the administration of either composition by itself.
  • dendritic atrophy is a prominent feature of neurodegenerative disease such as prion related diseases.
  • Increased Notch- 1 expression and cleavage releasing its intracellular domain (NICD) inhibit both dendrite growth and maturation. Knowing this relationship measurement where carried out on the levels of Notch- 1 and NICD in brains from mice inoculated with RML prions.
  • NICD The level of NICD was elevated in the neocortex while the level of ⁇ -catenin, which stimulates dendritic growth, was unchanged. During the incubation period, levels of the disease-causing prion protein isoform, PrP Sc , and NICD increased concomitantly in the neocortex. Additionally, increased levels of Notch- 1 mRNA and translocation of the NICD to the nucleus correlated well with regressive dendritic changes.
  • the present invention shows that PrP Sc in neurons and in ScN2a cells activates Notch- 1 cleavage, resulting in atrophy of dendrites in the CNS and shrinkage of processes on the surface of cultured cells. Accordingly, the present invention shows that diminishing Notch- 1 activation in vivo can prevent or even reverse neurodegeneration in various neurodegenerative diseases such as prion diseases.
  • the present invention shows that dendritic atrophy is accompanied by increased levels of the Notch- 1 intracellular domain (NICD).
  • NICD Notch- 1 intracellular domain
  • This was shown experimentally is neuronal nuclei of prion-infected mice. Elevated levels of NICD were also found in scrapie-infected neuroblastoma (ScN2a) but not in control, uninfected (N2a) cells. While long processes, also referred to as neurites, extend from the surface of N2a cells, ScN2a cells exhibit much shorter processes.
  • ScN2a cells were transfected with a Notch- 1 small interfering (si) RNA. The results showed that the normal, long-neurite phenotype was rescued.
  • Notch- 1 activation mediates dendritic atrophy in the brains of humans and animals with neurodegenerative diseases such as a prion disease.
  • mice were inoculated intrathalamically on the right with the RML strain of mouse prions, which produces clinical signs of disease at ⁇ 120 days postinoculation (dpi) and terminal disease at -150 dpi. They were sacrificed by decapitation at 30, 60, 90, 120, and 130-140 dpi. Age- and sex-matched controls were inoculated in parallel with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • N2a and ScN2a cells were maintained in Eagle's Minimum Essential Media with
  • N2a and ScN2a cells were plated onto glass coverslips that had been sequentially coated with 1 mg/ml poly-L-lysine in 0.1 M borate buffer, pH 8.5, and 0.1 mg mouse laminin/ml in PBS, respectively.
  • cells were grown in 6-well or 10-cm dishes coated with poly-lysine/laminin purchased from B-D Labware (Biocoat Cellware, Bedford, MA). To stimulate neural process formation, cells were then cultured in neurobasal medium containing the above-described antibiotics, 2 mM L- glutamine, N2 supplement, and 10 ng/ml murine 2.5 S nerve growth factor (Invitrogen, Carlsbad, CA).
  • the pools were suspended in 10 volumes (w/v) of an ice-cold solution containing 0.32 M sucrose in 5 mM Tris, pH 7.4, and homogenized in a Potter-El vehj em glass homogenizer with 14 strokes of a teflon pestle.
  • the crude homogenates were transferred to 50-ml conical tubes and centrifuged at 150Og for 10 min in a Beckman fixed rotor centrifuge to pellet cell debris and nuclei. The supernatants were removed and the pellet resuspended for a second slow-speed clarification process to obtain additional synaptosomes.
  • the supernatants from the two slow-speed centrifugations were pooled and centrifuged at 15,000g for 25 min in a fixed rotor KA-30 centrifuge (Optima LE- 8OK, Beckman, Fullerton, CA) to obtain a pellet enriched in synaptosomes. That pellet was resuspended in 10 volumes of RIPA lysis buffer (Ix PBS, pH 7.4; 1% Igepal CA-630; 0.5% sodium deoxycholate; 0.1% SDS), vortexed at 4 °C for 20 min, and at low speed in an Eppendorf tabletop centrifuge for 5 min to remove additional cellular debris from the synaptosomes.
  • RIPA lysis buffer Ix PBS, pH 7.4; 1% Igepal CA-630; 0.5% sodium deoxycholate; 0.1% SDS
  • PrP Sc For additional enrichment of PrP Sc , the final synaptosomal supernatants were subjected to two rounds of incubation with sodium phosphotungstate (NaPTA) to selectively precipitate PrP Sc (Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. & Prusiner, S. B. (1998) Nature Med. 4, 1157-1165; Safar, J.
  • the pellets were resuspended in 0.25 ml of PBS with 0.1%
  • PK proteinase K
  • PK digestion was stopped by the addition of 2 mM phenylmethylsulfonyl fluoride.
  • NaPTA was added to a final concentration of 0.32% followed by incubation at 37 °C for 1 h on a rocking platform and then centrifugation at 14,000g for 30 min at RT to precipitate protease-resistant PrP Sc .
  • the pellets were resuspended in equal volumes of SDS sample buffer and their proteins separated on 4— 12% Bis-Tris polyacrylamide gels and transferred to PVDF membranes for Western blot analysis recommended by the manufacturer (NuPage gel system, Invitrogen, Carlsbad, CA). The gels were loaded with aliquots containing equal amounts of protein based on the protein measurements made just before the NaPTA precipitation steps.
  • Neocortex dissected from mouse brains was homogenized in RIPA buffer.
  • cells were scraped from plates, pelleted at low speed, and lysed in 10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate; pH 7.5.
  • Protein concentrations were determined using the BCA protein assay using bovine serum albumin as a standard (Pierce, Rockford, IL).
  • N2a and ScN2a cells grown on coverslips were fixed for 10 min with 4% formaldehyde in PBS and then in 1:1 methanol/acetone for 10 min before being stained for mouse anti-neurofilament protein (NF200, Novacastra, Newcastle, UK), which was detected with anti-mouse Alexa Fluor 568. Fluorescence images were recorded with a Zeiss LSM 510 confocal microscope. Dendrite length and spine density
  • the primary apical and total basal dendrite lengths of neurons in layer 4 of the somatosensory neocortex overlying the hippocampus were measured at 200 ⁇ magnification.
  • Scion Image for Windows, release beta 4.0.2 (Scion Corporation, Frederick, MD), was the morphometric program used to make the measurements of dendrite parameters.
  • PCR reaction A total of 10 ⁇ l of cDNA was used for each PCR reaction.
  • Taqman probes and primers were designed using Primer 3 software (Whitehead Institute Center for Genome Research, Cambridge, MA) and were purchased through Integrated DNA Technologies (Coralville, IA).
  • the PCR reactions were carried out using Taqman core PCR reagents (Applied Biosystems) with 200 nM concentration of primers and 100 nM of fluorescent probe.
  • the Applied Biosystems HT 9700 sequence detector was programmed to an initial step at 50 0 C for 2 min and by 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min.
  • each sample was given a Cj value, which is defined as the number of PCR cycles needed to exceed a minimum fluorescent detection threshold.
  • Cj value is defined as the number of PCR cycles needed to exceed a minimum fluorescent detection threshold.
  • the analysis of the Taqman data was done using the comparative C T method (Applied Biosystems' Sequence Detection System User Bulletin #2). In each case, GAPDH was used as an endogenous reference.
  • siRNAs (Notch- 1 - 1 , Notch- 1 -2, and Notch- 1-3) against murine Notch- 1 were purchased from Ambion (Austin, TX). These siRNAs were transiently transfected into N2a and ScN2a cells using Lipofectamine 2000 per the manufacturer's instructions (Invitrogen, Carlsbad, CA). An siRNA targeted to the green fluorescent protein (GPF-22) was purchased from Qiagen (Valencia, CA) as a negative control in transfection experiments. Briefly, cells were plated the day before transfection at the desired density in maintenance media without antibiotics, typically 5000 cells/cm 2 . Cells were exposed to the siRNA-Lipofectamine mixture for 5 h before the maintenance medium was replaced by differentiation medium for the times indicated.
  • NICD levels increase during prion infection
  • Figure IA shows a Western blot analysis of NICD, ⁇ -catenin, and GAPDH in neocortical homogenates from four ill mice at 130 dpi and four age-matched, control mice.
  • Figure IB shows densitometry estimates of the concentrations of NICD and ⁇ -catenin relative to GAPDH show a statistically significant 2.5-fold increase in NICD concentration (Student t- test, * p ⁇ 0.0001) but no significant change in ⁇ -catenin during RML infection (filled bars) compared to controls (open bars).
  • Figure 1C is a confocal microscopy image which shows accumulation of NICD in the nucleus of neurons in RML-infected mice at 140 dpi. In uninfected control neurons, merge shows small amounts of NICD mostly in the cytoplasm.
  • a C-terminal Notch- 1 antibody was used to localize NICD.
  • a NeuN antibody was used to identify neurons.
  • DAPI identifies nuclei.
  • PrP Sc was enhanced by selective precipitation with NaPTA (Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. & Prusiner, S. B. (1998) Nature Med. 4, 1157-1165).
  • NaPTA NaPTA
  • the level of NICD in prion-inoculated mice increased progressively throughout the incubation period, reaching statistically significant increases at 120 and 130 dpi (Fig. 2a).
  • the levels of PrP Sc rose ⁇ 700-fold during the incubation period whereas the levels of NICD increased ⁇ 2.5-fold compared to controls.
  • FIG. 2a shows the kinetics of the log of neocortical PrP Sc accumulation in synaptosomes (filled squares) relative to NICD concentrations (filled circles) during the course of prion disease.
  • NICD levels in age-matched, PB S -inoculated mice are shown as controls (open circles).
  • Figure 2B is a plot of synaptosomal PrP So versus NICD shows a high degree of correlation.
  • Figure 2C shows qRT-PCR measurements of Notch- 1 mRNA levels at three time points during the course of prion disease (filled circles) relative to PBS-inoculated controls (open circles).
  • FIG. 2D and 2E Camera lucida drawings of Golgi-silver stained dendritic trees in Figures 2D and 2E show one type of regressive change, compare PBS-inoculated, age-matched control cerebral cortex (2D) to RML-infected cerebral cortex at 90 dpi (2E). Note that the Golgi method only stains a small percentage of neurons.
  • N2a cells (Fig. 3a). Both the ScN2a and control N2a cells were grown in a defined neurobasal medium containing N2 supplement and 10 ng/ml of nerve growth factor that promotes neuronal differentiation and growth of neuritic processes. By convention, processes with a length of less than twice the cell diameter were designated “processes” and those with lengths more than twice the cell diameter were designated “neurites.” Uninfected N2a cells grew numerous, long neurites under these culture conditions (Fig. 3b) whereas most ScN2a cells grew short processes (Fig. 3c).
  • ScN2a cells transfected with GFP siRNA had surface neurites (Fig. 4, b and d); in contrast, -27% of ScN2a cells treated with Notch- 1 siRNA grew long neurites (Fig. 4, c and d). The number of N2a cells with neurites ranged from -26% at 24 h (day 1) to -40% at 72 h (Fig. 4d). The difference between Notch- 1 siRNA-transfected ScN2a cells and non-transfected N2a cells was not statistically significant at 72 h (Fig. 4d). These results show that decreasing NICD concentration in ScN2a cells by selectively knocking down Notch- 1 mRNA expression resulted in recovery of the normal neurite phenotype.
  • N2a and ScN2a cells rescues the long-neurite phenotype in ScN2a cells.
  • NICD levels in N2a and ScN2a cells are reduced 3 d after transfection with 5 nM Notch- 1 siRNA (Notch-1-1, Ambion, Austin, TX) as shown in Figure 4A; in contrast, 5 nM GFP siRNA had no apparent effect.
  • Phase contrast microscopy shows that ScN2a cells treated with Notch- 1 siRNA (4C) have longer processes than ScN2a cells treated with GFP siRNA (4B) 3 d post-transfection and growth in differentiating medium. Bar in panel c represents 80 ⁇ m and also applies to panel b.
  • NICD dendritic growth in the developing nervous system is inhibited by NICD
  • the results show that increased levels of NICD and its translocation to the nucleus is responsible for dendritic atrophy in prion diseases.
  • ScN2a cells were studied to determine if the relationship between PrP Sc and Notch- 1 in brains of prion-infected mice was recapitulated in cultured cells. Compared to uninfected controls, NICD was elevated —50% and the number of cells with long neurites was significantly reduced in ScN2a cells.
  • the RML prion strain was used in both the mouse and cultured cell studies described here. Notch- 1 activation should occur with other strains. Much evidence indicates that different strains of prions reflect distinct conformers of PrP Sc (Legname, G., Baskakov, I. V., Nguyen, H. O., Riesner, D., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. (2004) Science 305, 673-6; Bessen, R. A. & Marsh, R. F. (1994) J. Virol. 68, 7859-7868; Telling, G. C, Parchi, P., DeArmond, S.
  • Notch- 1 activation should operate in the same manner in the pathogenesis of human prion disease, including the inherited forms of these disorders.
  • Notch 1 and the ⁇ -amyloid precursor protein (APP) are both cleaved by ⁇ -secretase (Lleo, A., Berezovska, O., Ramdya, P., Fukumoto, H., Raju, S., Shah, T. & Hyman, B. T. (2003) J Biol Chem 278, 47370-5). Cleavage of APP by ⁇ -secretase is a critical step in the formation of A ⁇ 42 peptide that comprises amyloid plaques in Alzheimer's disease (AD). Additionally, elevated levels of Notch- 1 protein (Berezovska, O., Xia, M. Q. & Hyman, B. T. (1998) J.
  • Notch- 1 The accumulation of the abnormal prion protein, PrP Sc , in neuronal plasma membranes triggers activation of Notch- 1.
  • This activation results in release of the Notch- 1 intracellular domain, designated NICD, and transport of NICD to the neuronal nucleus (Ishikura, N., Clever, J., Bouzamondo-Bernstein, E., Samayoa, E., Prusiner, S. B., Huang, E., and DeArmond, S. J. (2005).
  • Notch- 1 activation and dendritic atrophy in prion diseases PNAS
  • Notch- 1 activation may be specific for prion diseases; however, that remains to be verified.
  • Abnormal PrPSc can be formed in peripheral lymphocytes as well as in brain neurons during the course of Creutzfeldt- Jakob disease (CJD) in humans.
  • CJD Creutzfeldt- Jakob disease
  • Stanley Prusiner's laboratory has detected small amounts of PrPSc in the buffy coat from the blood of CJD patients using the CDI assay. Lymphocytes also express Notch- 1.
  • the desired cell concentration is 500-1500 cells/ ⁇ l. 400-800 ⁇ l of this suspension is added to Statspin 1-well cell concentrators with poly-L-lysine coated slides. A drop of 30% BSA can be added to promote cell adhesion. This assembly is placed in a Statspin Cytofuge-2 and spun at 1000 RPM for 4 min. The supernatant is then aspirated and the cell concentrator assembly is taken apart and the slides are immediately placed in 95% Methanol for 10 min. The slides are then placed in a PBS-Tween (0.05%) (PBST) wash to permeabilize the cells. The cells are ready for Immunofluorescent staining.
  • PBST PBS-Tween
  • Non-specific staining is blocked by incubating the cells with 10% normal donkey serum diluted in PBST for 20 min. Cells are then incubated for 60 min at RT in primary NICD-specific antibody (Notch-1 C-20, Santa Cruz Biotechnology) diluted 1 :50 diluted in 1% donkey serum. The slides are then washed several times in PBST. The secondary antibody (Donkey anti-goat linked with Alexafluor 488 dye) diluted 1 : 100 is added to the slides and allowed to incubate for 20min at RT. The slides are then washed again and then cover-slipped in a hard-set fluorescent mounting medium with DAPI to stain nuclei (Vector Laboratories). The intensity and subcellular distribution of the immunofluorescent staining of the slides are evaluated with a fluorescent microscope (See Figure 5).
  • NICD in CJD patients was located in the nuclei of lymphocytes whereas little or none was found in the nuclei of control lymphocytes.

Abstract

L'invention concerne un dosage permettant de déterminer les niveaux de Notch-1 dans un échantillon. Une augmentation des niveaux de Notch-1 au-delà des niveaux normaux est associée à une plus grande probabilité de la présence d'une maladie neurodégénérative. Le domaine intracellulaire de Notch-1 (NICD) peut être détecté à l'aide d'un anticorps spécifique au NICD. Un anticorps étiqueté permet de visualiser la quantité et le modèle de distribution du NICD qui est comparé à un témoin normal pour indiquer la présence d'une maladie neurodégénérative, y compris la maladie de Creutzfeldt-Jakob (CJD).
PCT/US2005/040625 2004-11-05 2005-11-02 Dosage de notch-1 utilise dans la detection de maladies neurodegeneratives WO2006053063A2 (fr)

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US9090690B2 (en) 2009-06-18 2015-07-28 Pfizer Inc. Anti Notch-1 antibodies
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US9499613B2 (en) 2008-07-08 2016-11-22 Oncomed Pharmaceuticals, Inc. Notch1 receptor binding agents and methods of use thereof
US9617340B2 (en) 2007-01-24 2017-04-11 Oncomed Pharmaceuticals, Inc. Compositions and methods for diagnosing and treating cancer
US9676865B2 (en) 2006-06-13 2017-06-13 Oncomed Pharmaceuticals, Inc. Antibodies to a non-ligand binding region of at least two NOTCH receptors

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US9676865B2 (en) 2006-06-13 2017-06-13 Oncomed Pharmaceuticals, Inc. Antibodies to a non-ligand binding region of at least two NOTCH receptors
US8513388B2 (en) 2006-10-19 2013-08-20 Genentech, Inc. Anti-Notch3 antibodies
US7915390B2 (en) 2006-10-19 2011-03-29 Genentech, Inc. Anti-Notch3 agonist antibodies and their use in the treatment of Notch3-related diseases
US7994285B2 (en) 2006-10-19 2011-08-09 Genentech, Inc. Anti-Notch3 antibodies
US9518124B2 (en) 2006-10-19 2016-12-13 Genentech, Inc. Anti-Notch3 agonist antibodies and their use in the treatment of Notch3-related diseases
US8187839B2 (en) 2006-10-19 2012-05-29 Genentech, Inc. Anti-notch3 agonist antibodies and their use in the treatment of notch3-related diseases
US8956811B2 (en) 2006-10-19 2015-02-17 Genentech Inc. Diagnosis of malignant neoplasms using anti-Notch3 antibodies
US8329868B2 (en) 2006-12-18 2012-12-11 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
US8148106B2 (en) 2006-12-18 2012-04-03 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
US9873734B2 (en) 2006-12-18 2018-01-23 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
US7935791B2 (en) 2006-12-18 2011-05-03 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
US9617340B2 (en) 2007-01-24 2017-04-11 Oncomed Pharmaceuticals, Inc. Compositions and methods for diagnosing and treating cancer
RU2476443C2 (ru) * 2007-06-04 2013-02-27 Дженентек, Инк. АНТИТЕЛА ПРОТИВ NRR Notch1 И СПОСОБЫ ИХ ПРИМЕНЕНИЯ
US8846871B2 (en) 2007-06-04 2014-09-30 Genentech, Inc. Anti-Notch1 NRR antibodies
WO2008150525A1 (fr) * 2007-06-04 2008-12-11 Genentech, Inc. Anticorps anti-notch 1 spécifiques à nrr et ses procédés d'utilisation
US10005844B2 (en) 2007-06-04 2018-06-26 Genentech, Inc. Polynucleotides encoding anti-Notch1 NRR antibody polypeptides
US9533042B2 (en) 2007-06-04 2017-01-03 Genentech, Inc. Anti-notch NRR antibodies and methods using same
US9499613B2 (en) 2008-07-08 2016-11-22 Oncomed Pharmaceuticals, Inc. Notch1 receptor binding agents and methods of use thereof
US9505832B2 (en) 2008-07-08 2016-11-29 Oncomed Pharmaceuticals, Inc. Method of treating cancer by administering a monoclonal antibody that binds human NOTCH2 and NOTCH3
US9132189B2 (en) 2008-07-08 2015-09-15 Oncomed Pharmaceuticals, Inc. Notch1 binding agents and methods of use thereof
US8404239B2 (en) 2008-10-01 2013-03-26 Genentech, Inc. Anti-Notch2 NRR antibodies
US9090690B2 (en) 2009-06-18 2015-07-28 Pfizer Inc. Anti Notch-1 antibodies
US9127060B2 (en) 2010-12-15 2015-09-08 Wyeth Llc Anti-Notch1 antibodies
US9433687B2 (en) 2012-11-07 2016-09-06 Pfizer Inc. Anti-Notch3 antibodies and antibody-drug conjugates

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