[go: up one dir, main page]

WO2018227212A1 - Signature génétique et biomarqueurs pour la sla et la démence - Google Patents

Signature génétique et biomarqueurs pour la sla et la démence Download PDF

Info

Publication number
WO2018227212A1
WO2018227212A1 PCT/US2018/036980 US2018036980W WO2018227212A1 WO 2018227212 A1 WO2018227212 A1 WO 2018227212A1 US 2018036980 W US2018036980 W US 2018036980W WO 2018227212 A1 WO2018227212 A1 WO 2018227212A1
Authority
WO
WIPO (PCT)
Prior art keywords
als
c90rf72
neuron
agent
poly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2018/036980
Other languages
English (en)
Inventor
Daniel A. MORDES
Kevin C. Eggan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
General Hospital Corp
Harvard University
Original Assignee
General Hospital Corp
Harvard University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by General Hospital Corp, Harvard University filed Critical General Hospital Corp
Publication of WO2018227212A1 publication Critical patent/WO2018227212A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • 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
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • ALS Amyotrophic lateral sclerosis
  • GGGGCC hexanucleotide
  • Carriers of the C90RF72 expansion can also present with frontotemporal dementia (FTD), which is characterized by frontotemporal lobar degeneration (FTLD) of the brain.
  • FTD frontotemporal dementia
  • FTLD frontotemporal lobar degeneration
  • these initially diverse diagnoses can progress toward the inclusion of neurological features from each condition, leading many to believe they are spectrums of the same disorder [5].
  • both diseases can be characterized by the presence of TDP -43 -positive inclusions [6].
  • nucleocytoplasmic transport [1, 5, 16].
  • the transcriptional response that occurs in various brain regions in ALS and FTD patients has the potential to provide useful insights into whether genetic subgroups of patients display common or divergent mechanisms, and for validating proposed mechanisms through which mutations act.
  • the present invention relates to the identification of a C90RF72 gene signature for ALS and methods of inducing the C90RF72 gene signature for ALS in neurons. Disclosed herein are methods of using the C90RF72 gene signature for screening for neurodegenerative disorders, diagnosing neurodegenerative disorders, and identifying agents for treating neurodegenerative disorders.
  • the invention disclosed herein relates, in some embodiments, to methods of inducing a C90RF72 gene signature (e.g., a C90RF72-ALS gene signature) in a neuron.
  • the method comprises contacting a neuron with an agent, thereby inducing the C90RF72 gene signature in the neuron.
  • the agent is a dipeptide repeat protein (DPR) (e.g., a synthetic DPR).
  • DPR dipeptide repeat protein
  • the DPR is selected from the group consisting of poly-GA, poly-GP, poly-GR, and combinations thereof.
  • the DPR is poly-GR.
  • the agent is selected from the group consisting of a proteasome inhibitor (e.g., MG-262 and/or thiostreptin), an HSP90 inhibitor (e.g., geldanamycin, monorden, alvespimycin, trichostatin A, tanespimycin, and/or parthenolide), and a translation inhibitor (e.g., puromycin).
  • a proteasome inhibitor e.g., MG-262 and/or thiostreptin
  • HSP90 inhibitor e.g., geldanamycin, monorden, alvespimycin, trichostatin A, tanespimycin, and/or parthenolide
  • the neuron is a human neuron (e.g., a stem cell-derived neuron). In some embodiments, the neuron is a non-human neuron.
  • the methods comprise
  • test agent (b) measuring level or activity of HSFl or one or more HSFl target genes in the contacted neuron; and (c) identifying the test agent as a candidate agent that modulates HSFl activation if the level or activity of HSFl or the one or more HSFl target genes in the neuron is decreased as compared with the level or activity of HSF l or the one or more HSFl target genes in the neuron that has not been contacted with the test agent.
  • the one or more HSFl target genes are selected from the group consisting of BAG2, CHORDC 1, CRYAB, DEDD2, DNAJB 1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and STIP1.
  • the methods comprise contacting a neuron with a proteasome inhibitor; contacting the proteasome inhibitor contacted neuron with a test agent; measuring activation of the HSFl pathway in the presence of the test agent; and identifying a candidate agent that inhibits HSF l pathway activation, wherein the test agent is the candidate agent for inhibiting HSF l pathway activation if the test agent decreases activation of the HSFl pathway in the proteasome inhibitor contacted neuron.
  • the methods comprise contacting a neuron with a poly-GR DPR; contacting the poly-GR DPR contacted neuron with a test agent; measuring activation of the HSF l pathway in the presence of the test agent; and identifying a candidate agent that inhibits HSFl pathway activation, wherein the test agent is the candidate agent for inhibiting HSF 1 pathway activation if the test agent decreases activation of the HSF 1 pathway in the poly-GR DPR contacted neuron.
  • a C90RF72 gene signature e.g., a C90RF72-ALS gene signature
  • the C90RF72 gene signature comprises increased expression of one or more genes selected from the group consisting of BAG2, CHORDC1, CRY B, DEDD2, DNAJB1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and STIP1.
  • kits for screening for ALS in a subject comprise screening a sample from the subject to detect a C90RF72 -ALS gene signature, wherein the presence of the C90RF72-ALS gene signature is indicative of the subject having ALS.
  • the methods comprise administering an effective amount of an agent that inhibits HSF1 pathway activation in the subject, thereby treating the neurodegenerative disorder.
  • inhibition of HSF 1 pathway activation comprises inhibition of one or more genes selected from the group consisting of BAG2, CHORDC1, CRYAB, DEDD2, DNAJB1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and STIP1.
  • the neurodegenerative disorder is a disorder that exhibits increased HSF1 pathway activation.
  • the neurodegenerative disorder is a disorder that exhibits a C90RF72 gene signature (e.g., is a C90RF72- associated neurodegenerative disorder).
  • the neurodegenerative disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), and combinations thereof.
  • the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS) or amyotrophic lateral sclerosis (ALS) combined with frontotemporal dementia (FTD) and/or frontotemporal lobar degeneration (FTLD).
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • FTLD frontotemporal lobar degeneration
  • Also disclosed herein are methods of treating a neurodegenerative disorder in a subject in need thereof
  • the agent inhibits the activity of one or more DPRs selected from the group consisting of poly-GA, poly-GP, and poly-GR. In certain embodiments the agent inhibits the activity of poly-GR DPR.
  • the neurodegenerative disorder is a disorder that exhibits increased HSF1 pathway activation. In some embodiments the
  • neurodegenerative disorder is a disorder that exhibits a C90RF72 gene signature (e.g., is a C9(9i? 72-associated neurodegenerative disorder).
  • the neurodegenerative disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), and combinations thereof.
  • the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS) or amyotrophic lateral sclerosis (ALS) combined with frontotemporal dementia (FTD) and/or frontotemporal lobar degeneration (FTLD).
  • FIGS. 1A-1G demonstrate identification of specific cellular pathways perturbed in sporadic ALS and C90RF72 -ALS.
  • FIG. 1A provides a diagram of RNA-seq datasets obtained from the frontal cortex and cerebellum by Prudencio et al.
  • FIG. IB shows a comparison of the significant (FDR ⁇ 0.05) differentially expressed transcripts in C90RF72-ALS (C9-ALS) and sporadic ALS (sALS). Note, there were no common transcripts between C90RF72-ALS and sporadic ALS in either brain region.
  • FIG. 1C shows a comparison of the differentially expressed transcripts by brain region.
  • FIG. 1A provides a diagram of RNA-seq datasets obtained from the frontal cortex and cerebellum by Prudencio et al.
  • FIG. IB shows a comparison of the significant (FDR ⁇ 0.05) differentially expressed transcripts in C90RF72-ALS (C9-ALS) and sporadic
  • FIG. 1G provides protein-protein interaction analysis of proteins encoded by the transcripts changed in C90RF72 -ALS revealed a protein chaperone network.
  • FIGS 2A-2B demonstrate activation of HSFl in C90RF72-ALS, FTD, and combined ALS/FTD patients.
  • FIG. 2B shows qRT-PCR for HSFl in the frontal cortex and cerebellum of these same cases.
  • FIGS. 3A-3D demonstrate DPRs induce expression of C90RF72 signature transcripts in human neurons.
  • FIG. 3 A provides a diagram of generation of human neurons from stem cells.
  • FIGS. 3C- 3D provide qRT-PCR of C9 ⁇ 9i? 72-chaperome transcripts (FIG. 3C) and HSFl (FIG.
  • FIGS. 4A-4G demonstrate detection of C9ftKF72-associated transcriptional changes in gain-of-function Drosophila models.
  • FIG. 4 A shows control UAS- (G4C2)8 and expanded UAS-(G4C2)49 transgenes were expressed in the adult fly nervous system using the drug-inducible Gal4 driver, elavGS, for 16d.
  • Quantitative PCR (qPCR) analysis of endogenous dHSFl and HSFl -regulated genes revealed significant upregulation with (G4C2)49 expression compared to (G4C2)8 controls. Differences in expression are likely underestimated as the analyses include neuronal and non-neuronal tissue while (G4C2)n was expressed only in neurons.
  • FIG. 4 A shows control UAS- (G4C2)8 and expanded UAS-(G4C2)49 transgenes were expressed in the adult fly nervous system using the drug-inducible Gal4 driver, elavGS, for 16d.
  • FIG. 4B provides qPCR analysis of a dHSFl overexpression mutant fly line shows endogenous HSF is upregulated approximately 2-fold in mutant flies compared to control.
  • FIG. 4C shows Western immunoblot analysis of expression of a control UAS-LacZ transgene confirmed that the HSF OE mutant did not affect the Gal4 UAS expression system.
  • FIG. 4D shows (G4C2)49 was expressed in the optic system of control animals or HSF OE animals using Gmr-Gal4. (G4C2)49 causes toxicity seen by pigment loss, reduced eye size, and disruptions in the normal ommatidial organization.
  • FIG. 4F provides Gmr-GAL4 driven expression of (GR)36 shows toxicity in control scenarios like (G4C2)49. HSF OE in these animals also causes enhanced toxicity (increased pigment loss, increased disruption of ommatidial organization, and reduced eye size).
  • FIGS. 5A-5F demonstrate bioinformatic method comparison for gene expression analysis of C90RF72-associated ALS and sporadic ALS (sALS) in the frontal cortex and cerebellum.
  • FIGS. 5A-5D provide gene density plots comparing the expression levels of differentially expressed transcripts as determined by the prior double cut-off method (
  • FIG. 5E provides Venn diagrams demonstrating considerable differences in the designated disease-associated transcripts between these bioinformatics methods in both brain regions.
  • FIG. 5F provides table of the number of differentially expressed transcripts as determined by Prudencio et al., the double cut-off method and the FDR method used here.
  • FIGS. 6A-6B demonstrate gene networks in C90RF72-ALS and sporadic ALS.
  • FIG. 6A shows protein-protein interaction network derived from differentially expressed transcripts in C90RF72-ALS cerebellum. Those transcripts that are differentially expressed in both the frontal cortex and the cerebellum in C90RF72- ALS are highlighted by dashed yellow circles, and predominantly consist of heat shock proteins and protein chaperones.
  • FIG. 6B shows protein-protein interaction network derived from differentially expressed transcripts in the sporadic ALS cortex.
  • FIGS. 7A-7B demonstrate activation of HSF1 in C90RF72-ALS, FTLD, and combined ALS/FTLD patients.
  • FIG. 7B shows correlation of HSF1 levels and HSF1 target gene levels in the frontal cortex and cerebellum in C90RF72-ALS/FTLD. Spearman's R 2 values are plotted for each target gene, error bars denote 95% confidence interval, p-value ⁇ 0.0001 in all cases.
  • FIG. 8 demonstrates poly-GR expression results in the upregulation of heat shock response genes and dHSFl in the adult fly nervous system.
  • UAS-(GR)36 was expressed in the adult fly nervous system using the drug-inducible Gal4 driver, elavGS, for 16d.
  • qPCR analysis of endogenous HSF1 -regulated genes and dHSFl revealed significant upregulation of the Drosophila orthologs of many of the genes identified in patient studies, suggesting that poly-GR is contributing to the altered transcriptome in C90RF72-ALS FTLD patients.
  • FIG. 9 provides external eye quantification scale for (GR)36 animals.
  • (GR)36 animals received a score between 0 (normal eye) and 11 (extreme toxicity causing lethality).
  • Gmr-GAL4 > (GR)36 animals receive a score between 5-6.
  • FIGS. 10A-10I demonstrate gene expression analysis of C90RF72-associated ALS (C9-ALS) and sporadic ALS (sALS) patients.
  • FIG. 10A provides a diagram of RNA-seq datasets grouped by disease state and bioinformatics analysis and a Table of the number of brain samples used for RNA-seq obtained from the frontal cortex and cerebellum by Prudencio et al.
  • FIGS. 10B and IOC provide gene density plots comparing the expression levels of significantly changed transcripts as determined by the double cut-off method (FIG. 10B) and FDR method (FIG. IOC) compared to the distribution of all transcripts.
  • FIG. 10B double cut-off method
  • FDR method FDR method
  • FIG. 10D provides Venn diagrams comparing the number of differentially expressed transcripts as determined by double cut-off method and the FDR method based on disease state. Note no significantly changed transcript were detected in the cerebellum of C90RF72-ALS by the FDR method.
  • FIG. 10E provides Venn diagrams comparing the differentially expressed transcripts of C90RF72-ALS (C9-ALS) and sporadic ALS (sALS). Note there was no common transcripts between C90RF72-ALS and sporadic ALS in either brain region based on the FDR method.
  • FIGS. 10F and 10G show gene ontology (GO) term biological process clusters based on FDR method differentially expressed transcript from the cortex of C90RF72-ALS and sporadic ALS.
  • FIG. 10H shows protein-protein interaction network of differentially expressed transcripts from C9-ALS cortex based on the FDR method.
  • FIG. 101 provides correlation of the log2 fold change (FC) gene expression of transcripts that were significantly changed (FDR ⁇ 0.05) in both the cortex and the cerebellum in C90RF72-ALS.
  • FIGS. 11A-11D demonstrate activation of HSFl in C90RF72-ALS/FTO.
  • FIGS. 11 A and 1 IB provide quantitative RT-PCR of HSFl levels in the frontal cortex (FIG. 11 A) and cerebellum (FIG. 1 IB) from samples of patients with ALS and/or FTD harboring mutations in C90RF72 vs. ALS and/or FTD patients without C90RF72 mutations and controls.
  • FIG. 11C provides representative
  • FIG. 1 ID shows qPCR of HSFl targets genes in the cerebellum of C90RF72 (C9) or sporadic (non-C9) ALS and/or FTD cases and controls cases.
  • FIGS. 12A-12E demonstrate that the connectivity map connects the
  • FIG 12A provides a diagram of connectivity map analysis.
  • FIG. 12B provides volcano plot of compounds associated with the C90RF2-ALS cortex signature genes.
  • FIG. 12C provides volcano plot of compounds associated with the C90RF72-ALS cerebellum signature genes. Compounds found in both sets are highlighted in bold.
  • FIG. 12D shows connectivity map analysis yields hit compounds that perturb proteostasis through various mechanisms, as well as other compounds with their known target of action provided.
  • C90RF72-ALS connectivity scores are in parentheses.
  • FIG. 12E shows heat map of the connectivity scores of compounds that affect pathways implicated in ALS. Significantly connected compounds (p ⁇ 0.001) are highlighted with an asterisk.
  • FIGS. 13A-13D demonstrate inhibition of the proteasome recapitulates the C90RF72-ALS gene signature in neurons in vitro.
  • FIG. 13A provides schematic for the generation of purified neurons from stem cells and subsequent drug treatment with a proteasome inhibitor, MG132.
  • FIG. 13B shows gene ontology (GO) term biological process clusters based on transcripts induces by MG132 treatment in motor neurons, which is similar to the C90RF72-ALS GO term clusters.
  • FIG. 13C provides transcripts levels of all coding C90RF72-ALS signature genes. Genes that significantly changed in expression after proteasome inhibition at both doses are bolded (adjusted p-value ⁇ 0.0001).
  • FIG. 13A provides schematic for the generation of purified neurons from stem cells and subsequent drug treatment with a proteasome inhibitor, MG132.
  • FIG. 13B shows gene ontology (GO) term biological process clusters based on transcripts induces by MG132 treatment in motor neurons, which is similar to the C90RF72-
  • 13D shows correlation of log2 fold change in chaperome-related genes in C90RF72-ALS cortex, sporadic ALS (sALS) cortex, and C90RF72-ALS cerebellum compared to changes induced after proteasome inhibition in motor neurons in vitro. Linear regression and R2 values are displayed.
  • FIGS. 14A-14F demonstrate activation of the HSF1 pathway by C90RF72 hexanucleotide repeat expansion-associated dipeptide repeat proteins.
  • FIG. 14A shows cell viability in stem cells following synthetic dipeptide repeat protein (DPR) treatment. Note GAPR is a scrambled control peptide. Cell viability in neurons following DPR treatment.
  • FIG. 14B shows mislocalization of TDP-43 after DPR treatment. Correlation between DAPI (nuclear) and TDP-43 immunofluorescence in motor neurons following DPR treatment.
  • FIG. 14C provides quantitative RT-PCR of heat-shock related C9-ALS signature genes in neurons following DPR treatment. Normalization is performed to the geometric mean of GAPDH and actin.
  • FIG. 14A shows cell viability in stem cells following synthetic dipeptide repeat protein (DPR) treatment. Note GAPR is a scrambled control peptide. Cell viability in neurons following DPR treatment.
  • FIG. 14B shows mislocalization of TDP-43
  • FIG. 14D shows correlation between log2 fold changes of tested C9-ALS signature genes in poly- PR treated neurons and log2 fold changes in C9-ALS frontal cortex.
  • FIG. 14E provides a model of neuron proteostasis in healthy neurons.
  • FIG. 14F shows putative mechanisms of disruption of proteostasis in C90RF72-ALS/FTD neurons
  • the present invention relates to the characterization of a specific C90RF72 gene signature, specifically the C90RF72 gene signature with respect to ALS patients.
  • Disclosed herein are methods for inducing the C90RF72 gene signature in neurons, as well as methods of using the C90RF72 gene signature to screen for neurodegenerative disorders, diagnose neurodegenerative disorders, and identify agents for treating neurodegenerative disorders.
  • the inventions disclosed herein relate to a C90RF72 gene signature.
  • the C90RF72 gene signature as described herein is a C90RF72 gene signature that may be identified in patients having amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and/or frontotemporal lobar degeneration (FTLD). FTD may be characterized by FTLD of the brain.
  • the C90RF72 gene transcript exhibits increased activation of HSF1, as well as increased expression of one or more HSF1 target genes.
  • the one or more HSF1 targets genes include BAG2, CHORDC1, CRYAB, DEDD2, DNAJB1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and/or STIP1.
  • the inventions disclosed herein relate to methods of inducing the C90RF72 gene signature, e.g., the C90RF72-ALS gene signature.
  • a C90RF72 gene signature is induced in a neuron.
  • the C90RF72 gene signature may be induced by contacting the neuron with an agent.
  • the neuron may be a human neuron or a non -human neuron.
  • the neuron is a human neuron, e.g., a stem cell-derived neuron.
  • the agent induces an HSF1 response, e.g., activation of HSF1.
  • the agent is a dipeptide repeat protein (DPR), and in some embodiments is a synthetic DPR.
  • DPR dipeptide repeat protein
  • the DPR may be selected from the group consisting of poly-glycine-arginine (poly-GR), poly-glycine-alanine (poly-GA), poly- glycine-proline (poly-GP), and combinations thereof.
  • a C90RF72 gene signature is induced in a neuron by contacting the neuron with poly- GR DPR.
  • a C90RF72 gene signature is induced in a neuron by contacting the neuron with poly-GA DPR.
  • a C90RF72 gene signature is induced in a neuron by contacting the neuron with poly-GP DPR.
  • treatment of a neuron with one or more DPRs results in increased expression of one or more C90i? 77-chaperome transcripts (e.g., GAPDH, ACTIN, SERPINH1, STIP1, BAG3, CHORDC 1, HSPA1B, and HSPA6) and/or increased expression of HSF 1.
  • C90i? 77-chaperome transcripts e.g
  • the agent is selected from the group consisting of a proteasome inhibitor, an HSP90 inhibitor, a translation inhibitor, and combinations thereof.
  • the agent is a proteasome inhibitor (e.g., MG-132, MG-262, or thiostreptin).
  • the agent is a translation inhibitor (e.g., puromycin).
  • the agent is an HSP90 inhibitor (e.g., geldanamycin, monorden/radcicol, alvespimycin/17-DMAG, trichostatin A, tanespimycin/17-AAG, or parthenolide).
  • the agent is selected from the group consisting of 15-delta prostaglandin J2, securinine, astemizole, thioridazine, and nordihydroguaiaretic acid. In some aspects the agent is any combination of agents disclosed herein. In some aspects treatment of a neuron with one or more proteasome inhibitors results in increased expression of one or more C90RF71- chaperome transcripts (e.g., DNAJB4, CHORDC1, SERPINHl, BAG3, FKBP4, STIP1, HSPA6, HSPB 1, HSPA1B, DNAJB 1, and HSPA1A).
  • C90RF71- chaperome transcripts e.g., DNAJB4, CHORDC1, SERPINHl, BAG3, FKBP4, STIP1, HSPA6, HSPB 1, HSPA1B, DNAJB 1, and HSPA1A.
  • the inventions disclosed herein relate to methods of screening subjects or patients for a C90RF72 gene signature.
  • methods include detecting a C90RF72 gene signature in a subject by obtaining a sample from the subject and screening the sample to detect if the gene signature is present in the sample.
  • the C90RF72 gene signature is a C90RF72- ALS gene signature.
  • the C90RF72 gene signature exhibits increased expression of one or more genes selected from the group consisting of BAG2, CHORDC1, CRYAB, DEDD2, DNAJB1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and STIP1.
  • the C90RF72 gene signature exhibits increased activity of HSFl . Detection of a C90RF72 gene signature in a patient may be indicative of the patient having ALS, FTD, and/or FTLD.
  • a method of screening for ALS in a subject comprises screening a sample from the subject to detect a C90RF72 -ALS gene signature, wherein the presence of the gene signature is indicative of the patient having ALS.
  • the patient may further exhibit symptoms of FTD and/or FTLD.
  • the inventions disclosed herein relate to methods of screening one or more test agents to identify candidate agents which modulate HSF l activation in a cell.
  • “Modulate” as used herein means to decrease (e.g., inhibit, reduce) or increase (e.g., stimulate, activate) a level, response, property, activity, pathway, or process.
  • a “modulator” is an agent capable of modulating a level, response, property, activity, pathway, or process. In some aspects a modulator is an inhibitor or blocker of HSFl activation.
  • agent as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc.
  • an agent can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non- proteinaceous entities.
  • an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof.
  • a method of screening comprises (a) contacting a neuron (e.g., a stem cell-derived neuron) exhibiting a C90RF72-ALS gene signature with a test agent; (b) measuring level or activity of HSFl or one or more HSF l target genes in the contacted neuron; and (c) identifying the test agent as a candidate agent that modulates HSF l activation if the level or activity of HSF l or the one or more HSF l target genes in the neuron is decreased as compared with the level or activity of HSF l or the one or more HSFl target genes in the neuron that has not been contacted with the test agent.
  • a neuron e.g., a stem cell-derived neuron
  • An HSFl target gene may be selected from the group consisting of BAG2, CHORDC1, CRYAB, DEDD2, DNAJB 1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, DEDD2, and STIP1.
  • a method of identifying a candidate agent that inhibits HSFl pathway activation comprises: (a) contacting a neuron (e.g., a stem cell-derived neuron) with a proteasome inhibitor; (b) contacting the proteasome inhibitor contacted neuron with a test agent; (c) measuring activation of the HSFl pathway in the presence of the test agent; and (d) identifying a candidate agent that inhibits HSFl pathway activation, wherein the test agent is the candidate agent for inhibiting HSFl pathway activation if the test agent decreases activation of the HSFl pathway in the proteasome inhibitor contacted neuron.
  • a neuron e.g., a stem cell-derived neuron
  • a method of identifying a candidate agent that inhibits HSFl pathway activation comprises (a) contacting a neuron (e.g., a stem cell-derived neuron) with a dipeptide repeat protein (e.g., poly-GR DPR); (b) contacting the DPR contacted neuron with a test agent; (c) measuring activation of the HSFl pathway in the presence of the test agent; and (d) identifying a candidate agent that inhibits HSFl pathway activation, wherein the test agent is the candidate agent for inhibiting HSFl pathway activation if the test agent decreases activation of the HSFl pathway in the DPR contacted neuron.
  • a neuron e.g., a stem cell-derived neuron
  • a dipeptide repeat protein e.g., poly-GR DPR
  • the present invention also provides a method for disrupting HSFl activation.
  • HSFl activation is disrupted by genomic modification (e.g., using CRISPR/Cas or TALEN systems).
  • CRISPR/Cas systems can employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; l(6)e60).
  • the CRISPR/Cas system is a CRISPR type I system.
  • the CRISPR/Cas system is a CRISPR type I system.
  • CRISPR/Cas system is a CRISPR type II system. In some embodiments, the CRISPR/Cas system is a CRISPR type V system.
  • the inventions disclosed herein relate to methods of treating neurodegenerative disorders.
  • methods of treating a neurodegenerative disorder comprise blocking or inhibiting activation of the HSFl pathway.
  • activation of the HSFl pathway is disrupted using genome editing (e.g., CRISPR/Cas).
  • a method of treating a neurodegenerative disorder comprises administering an effective amount of an agent to a subject, wherein the agent inhibits or blocks HSF 1 pathway activation (e.g., in a neuron).
  • the agent inhibits DPR activation of the HSF1 pathway.
  • blocking HSF 1 pathway activation or inactivating the HSF 1 pathway results in inhibiting (e.g., decreasing expression of) one or more HSF1 target genes.
  • the HSF 1 target genes may be selected from the group consisting of BAG2, CHORDC1, CRYAB, DEDD2, DNAJB1, DNAJB4, FKBP4, HSPA1A, HSPA1B, HSPB1, SERPINH1, and STIP1.
  • the agent inhibits activation of the HSF 1 pathway by poly-GA DPR, poly-GP DPR, and/or poly-GR DPR.
  • the neurodegenerative disorder is a disorder that exhibits increased HSF1 pathway activation.
  • the neurodegenerative disorder is a disorder that exhibits a C90RF72gene signature (e.g., is a C90RF72- associated neurodegenerative disorder).
  • the neurodegenerative disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), and combinations thereof.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • FTLD frontotemporal lobar degeneration
  • the neurodegenerative disorder is ALS.
  • the neurodegenerative disorder is ALS in combination with FTD and/or FTLD.
  • the agents disclosed herein can be provided in pharmaceutically acceptable compositions.
  • These pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the agents, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), gavages, lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally;
  • oral administration for example,
  • agents can be implanted into a patient or injected using a drug delivery system.
  • a drug delivery system See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and
  • the term "pharmaceutically acceptable” refers to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term "pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body (e.g., to one or more muscle cells or satellite cells).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body (e.g., to one or more muscle cells or satellite cells).
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • excipient e.g., pharmaceutically acceptable carrier or the like are used interchangeably herein.
  • terapéuticaally-effective amount means that amount of an agent, material, or composition comprising an agent described herein which is effective for producing some desired therapeutic effect in at least a sub- population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • an amount of an agent administered to a subject that is sufficient to produce a statistically significant, measurable decrease in the activation of HSFl .
  • a therapeutically effective amount of the agents and compositions disclosed herein is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject' s history, age, condition, sex, and the administration of other pharmaceutically active agents.
  • administer refers to the placement of an agent or composition into a subject (e.g., a subject in need) by a method or route which results in at least partial localization of the agent or composition at a desired site such that desired effect is produced.
  • Routes of administration suitable for the methods of the invention include both local and systemic routes of administration. Generally, local administration results in more of the administered agents being delivered to a specific location as compared to the entire body of the subject, whereas, systemic
  • administration results in delivery of the agents to essentially the entire body of the subject.
  • compositions and agents disclosed herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • oral or parenteral routes including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • the compositions are administered by intravenous infusion or injection.
  • a "subject” means a human or animal (e.g., a mammal).
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • a primate e.g., a human.
  • subject can be male or female.
  • the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • GGGGCC hexanucleotide repeat expansion in C90RF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS) and
  • FTLD frontotemporal lobar degeneration
  • RNA- sequencing datasets were generated from the frontal cortex as well as the cerebellum of sporadic ALS cases, C90RF72-ALS cases and controls [17].
  • Prudencio et al. a "double-cutoff method" was used for identifying genes whose expression was significantly changed in each class of ALS patient relative to controls (methods). Although such methods are useful for identifying changes in gene expression, they tend to be more sensitive to large fold-changes in less abundant transcripts, while modest fold-changes in abundant transcripts may go undetected (FIG.
  • C90RF72 ALS patient classes [17].
  • the cortex contains distinct p62-positive DPR neuronal inclusions, and the cerebellum is characterized by abundant DPR inclusions [11, 17, 20, 21].
  • identifying transcripts with expression changes shared in both the frontal cortex and the cerebellum might lead to genes and pathways that were reproducibly induced by the C90RF72 repeat expansion.
  • transcripts significantly changed in C90RF72 and sporadic ALS cortex pointed to particular pathways that might be responding to disease processes
  • gene ontology (GO) analysis was employed.
  • differentially expressed transcripts detected in sporadic ALS were associated with functions in the mitochondrial respiratory chain complex assembly (8.53 * 10 "21 ) and related terms (FIG. IF), and included 9 members of the NADH dehydrogenase (complex I) enzyme and 6 components of cytochrome oxidase C (complex IV) (FIG. 6B).
  • HSF 1 transcription factor heat shock factor 1
  • HSF1 expression in these same samples demonstrated that it was significantly more abundant in both the cortex and cerebellum relative to sporadic ALS cases (P ⁇ 0.05) (FIG. 2B).
  • a strong and consistent correlation was found between the levels of HSF1 and each of these C90RF72 -chaperome transcripts in both brain regions (p ⁇ 0.0001 for each gene, FIG. 7B).
  • the relationship between the transcript levels of HSF1 and HSPB1 yielded an R 2 value of 0.73 (95% CI 0.63 - 0.81) in the cortex and 0.65 (95% CI 0.52 - 0.75) in the cerebellum.
  • DPRs are sufficient to induce a C90R 72-associated transcriptional changes
  • the C90RF72 GGGGCC repeat expansion is translated from both sense and anti-sense transcripts through non-ATG translation to generate 5 distinct dipeptide repeat proteins (DPRs), e.g. poly-glycine-arginine (poly-GR) [5, 11, 29]. It was considered if DPRs alone were sufficient to induce the upregulation of C90RF72 signature transcripts. Therefore, the effects of synthetic DPRs were tested in human stem cell-derived neurons [13]. GP 10 , GAi 0 and a scrambled GAPR 5 control were not acutely toxic to the parental human stem cell line or stem-cell derived neurons.
  • DPRs dipeptide repeat proteins
  • poly-GR poly-glycine-arginine
  • poly-GRio resulted in a dose-dependent decrease in the viability of stem cell- derived neurons, but not the stem cell from which they were produced (FIG. 3B).
  • poly-GA and poly-GR led to the significant upregulation oiHSPAlB (p ⁇ 0.01), as well as additional C90RF72 signature transcripts (FIG. 3C).
  • poly-GA is not associated with decreased viability in these conditions, this suggests that the observed transcriptional changes are not simply a consequence of general neuronal toxicity.
  • R 2 0.58
  • a Drosophila gain-of-function transgenic model engineered to express 49 pure GGGGCC repeats driven by a drug-inducible neuronal-specific ElavGS-GAL4 driver was evaluated [14, 30].
  • Fly models expressing toxic GGGGCC repeats produce DPRs and RNA foci [14, 31, 32].
  • Significant increased expression of the Drosophila orthologs of conserved C9(9i? 72-associated HSPs and protein chaperones was found in flies expressing 49 repeats in neurons compared to controls (FIG. 4A). Upregulation of HSFl -associated genes was observed in the absence of significant animal death, arguing that the effect is specific to expression of
  • HSF l Activation of the HSF l pathway has been proposed to be protective in several neurodegenerative diseases associated with protein aggregation as a means to combat the cellular effects of toxic proteins [33].
  • HSF l may be a potential modifier of C90RF72 gain-of-function toxicity.
  • dHSFl Drosophila HSFl ortholog
  • arginine-rich DPRs are particularly toxic in model systems, including Drosophila [14].
  • GGGGCC 49 is associated with the production of both DPRs and potentially toxic RNA
  • the transcriptional effects of poly-GR in vivo were assayed.
  • the effects of modulating dHSFl levels in the optic system of poly-GR Drosophila was tested again using Gmr-GAL4 to drive transgene expression.
  • HSFl pathway is highly conserved from budding yeast to mammals and is an important mediator of the compensatory response to disruptions in proteostasis, such as heat shock [27]. Impairment of HSFl activity and loss of protein chaperone function have been reported to occur with ageing and in the setting of age- related neurodegeneration [33, 35, 36]. For instance, in models of poly-glutamine repeat-associated Huntington disease decreased expression of HSFl target genes is observed and may contribute to protein aggregation [37].
  • HSFl is generally not thought to be regulated at the transcriptional level in the context of neurodegeneration. Upregulation of HSFl itself in C90RF72- ALS/FTLD was found, as well as a strong correlation between levels of HSFl and its target genes.
  • HSFl is a protective factor that helps neurons cope with stress associated with misfolded proteins and protein aggregates [38].
  • Pharmacological activation of HSFl has been proposed as a therapeutic strategy to enhance protein chaperone function and neuronal survival in
  • arimoclomol which may act to enhance HSF l -pathway activation, has been shown to delay disease progression in an SOD1 overexpression mouse model [40].
  • a phase II clinical trial for arimoclomol was recently conducted for a subtype of familial ALS associated with mutations in SOD1 and was found to be well -tolerated [41].
  • Drosophila orthologs of the same genes that were upregulated in C90RF72 patient brains were found. This is consistent with the notion that more potent expression of DPRs in models is essential to recapitulate C90RF72 transcriptional changes and disease phenotypes.
  • the methods and findings described herein, starting with unbiased transcriptional analysis of patient samples, may be useful for the
  • the processed gene expression count matrix of the brain-derived RNA-seq datasets from Prudencio et al. were obtained via GEO (GSE67196).
  • the data was analyzed using the R library "edgeR” as described by Prudencio et. al., with modifications as follows [17, 48].
  • Statistical inference was performed with two methods which is referred to as “double cut-off and "FDR".
  • double cut-off method as described by Prudencio et al.
  • differentially expressed genes called by this approach had to pass two filters: one cut-off of absolute log2fold change > 2 and a second cut-off of unadjusted p-value ⁇ 0.05.
  • the false discovery rate was controlled using the Benjamini-Hochberg method [49] and all genes below a threshold FDR of 0.05 were considered to be significantly
  • retinoic acid Sigma Aldrich, ⁇ ⁇
  • smoothened agonist SAG, DNSK, ⁇
  • BMP inhibitor LDN-193189, DNSK, 100 nM
  • TGF-beta inhibitor SB431542, DNSK, lOuM
  • retinoic acid smoothened agonist
  • GSK3-beta inhibitor SU-5402, DNSK, 4 ⁇
  • gamma-secretase inhibitor DAPT, DNSK, 5uM
  • GFP-positive motor neuron was purified with FACS and then plated on poly-D- lysine(Sigma Aldrich)/laminin(Life Technologies)-coated plates and allowed to mature two weeks in media supplemented neurotrophic factors (BDNF, CNTF and GDNF). Upon completion of the differentiation protocol, cells were sorted via flow- cytometry for GFP-positive cells to yield GFP-positive neurons plated on
  • DPRs dipeptide repeat proteins
  • UAS-(G4C2)n or UAS-(GR)36 transgenes were driven by elavGS, a drug- inducible Gal4 driver that expresses only in neurons. Crosses were setup and maintained at 24°C.
  • Female progeny with the desired genotype were collected and matured to l -3d before being transferred to vials containing 40ug/ml of RU486. Animals were aged on RU486-infused food 16d while being flipped onto fresh drug- infused food every 2-days. Total RNA was collected from heads of frozen animals using Trizol, converted to cDNA using random primers, and analyzed by qPCR using SYBR Green.
  • (G4C2)49 expression causes an average degenerative score of 4-5 across multiple studies.
  • (GR)36 expression causes an average degenerative score of 5-6 across multiple studies.
  • crosses for (G4C2)n were setup and maintained at 24°C and (GR)36 at 21°C.
  • Male progeny with the desired genotype were collected daily and matured to l-2d before imaging on a Leica Apol6 microscope. Severity of the external eye phenotype was determined post-imaging while looking for changes in red pigmentation, ommatidial organization, and eye size.
  • Full genotypes for (G4C2)n are as follows: "Contror « wl 1 18;; UAS ⁇ (G4C2)n, Gmr ⁇ GaS4/+ and "HSF QE" ⁇ W1118;; UAS ⁇ (G4C2)a Gmr-Gal4/FISF[+t8].
  • DNAJB4 AGGTCGCAGAAGCTTATGAAGT CCTCCTGCTCCTCCTTTCA
  • HSFAIA CGGCAAGGTGGAGATCAT GGTGTTCTGCGGGTTCAG
  • HSPA1B AAGGGTGTTTCGTTCCCTTT TAGTGTTTTCGCCAAGCAAA
  • HSP6S GAAGGCACTCAAGGACGCTAAAATG CTGAACCTTGGGAATACGAGTG dH. ⁇ GGACAAGGATGCCAAGAAGAAGAAG CAGTCGTTGGTCAGGGATTTGTA (HSP83) G
  • iiHSP4 GAGATCATCAAGCCCACCACAAC CGGGAAACTTAATGTCGAAGGAG
  • Prudencio M., et al., Distinct brain transcriptome profiles in C9orf72- associated and sporadic ALS. Nat Neurosci, 2015. 18(8): p. 1 175-82.
  • Prudencio, M., et al., Repetitive element transcripts are elevated in the brain ofC9orp2 ALS/FTLD patients.
  • Example 2 both re-presents certain data from Example 1 and provides additional data.
  • Prudencio and colleagues reported the generation of RNA sequencing data from the frontal cortex and cerebellum of control individuals, as well as sporadic (sALS) and C90RF72 ALS cases (FIG. 10A).
  • sALS sporadic
  • C90RF72 ALS cases C90RF72 ALS cases
  • HSPA6 heat-shock protein
  • HSFl heat shock factor-1
  • HSFl transcription factor 1
  • HSFl heat shock factor-1
  • HSFl complexed with HSPs in an inactive state
  • HSFl Upon heat shock, HSFl is released and accumulates in the nucleus to induce the transcription of HSPs and other chaperones (28).
  • HSFl 11 chaperome-associated transcripts in the C90RF72-ALS gene signature were established target genes of HSF l, suggesting that activation of HSF l could be driving, at least a subset of, the transcriptional changes observed ((26), (29), (30)).
  • pathological alterations in HSFl could be detected in human brains with C90RF72-ALS. It was found that HSFl mRNA levels were upregulated specifically in C9(9i? 72-associated
  • FIG. 11A and 11B neurodegenerative disease compared to either controls or non-C90RF72 patients (p ⁇ 0.05)
  • histological analysis revealed increased nuclear HSF l protein expression in the cerebellar neurons of C90RF72 -ALS/FTD cases, most notably in Purkinje neurons, but not in controls (FIG. 11C).
  • RNA levels of each of the 12 HSF1 targets tested were significantly increased in C90RF72-ALS/FTO compared to sporadic cases (p ⁇ 0.01) (FIG. 11D) and all were significantly increased relative to controls (p ⁇ 0.05 or lower for each gene).
  • BAGS which encodes an HSP70 co-chaperone protein
  • C90RF72 -ALS FTD was markedly upregulated in C90RF72 -ALS FTD compared to controls (p ⁇ 0.01) and also compared to sporadic disease (p ⁇ 0.001) (FIG. 1 1D).
  • a significant correlation between HSF1 expression levels and the expression levels of each of these target genes in this cohort of ALS, FTD, and ALS FTD patients was found (p ⁇ 0.0001 for each gene).
  • the relationship between transcript levels of HSF1 and HSPA1A, encoding an HSP70 protein yielded an R2 value of 0.63 (95% CI 0.51 - 0.74).
  • MG-262 is a cell-permeable 26S proteasome inhibitor, and had near maximum connectivity scores with the C90RF72-ALS signature from both brain regions (0.957 cortex, 0.979 cerebellum).
  • Geldanamycin which is an antibiotic that inhibits the ATPase activity of HSP90 chaperone proteins, and its derivatives (e.g. tanespimycin/17-AAG) were also hits.
  • Puromycin is an aminonucleoside antibiotic that resembles tRNA and inhibits translation through premature chain termination, resulting in the production of truncated puromycyl-containing peptides.
  • Some compounds identified through connectivity analysis also had known off-target effects on these molecular targets. For instance, 15d-PGJ2 is an anti-inflammatory agent, but has been reported to induce aggregation of ubiquitinated proteins and inhibit the proteasome (32).
  • ribonucleoproteins have been implicated in ALS and FTD.
  • agents that globally alter transcription such as thioguanosine, were also not connected to either C90RF72- ALS or sALS gene expression changes (FIG. 12D).
  • C90RF72-ALS gene signature many compounds identified associated with the C90RF72-ALS gene signature were also associated with the gene expression profiles of proteasome- inhibited neurons. Overall, this analysis suggests that the disease-state present in C90RF72-ALS brain mimics the neuronal response in vitro to proteasome inhibition.
  • DPRs are sufficient to induce a heat shock-associated transcriptional response
  • the C90RF72 GGGGCC repeat expansion is translated in the sense and anti- sense direction through non-ATG translation to generate 5 distinct dipeptide repeat proteins (DPRs).
  • the sense DPRs (poly-GA, -GP, -GR) are however thought to be more abundant in patient brains (5, 10, 35). Given that DPRs may be in an intrinsically unfolded state, it was considered whether they interacted with protein chaperones in general, and the specific components of the chaperome network that were found upregulated in C90RF72-ALS. Recently, independent mass spectrometry efforts were conducted to identify DPR interacting proteins (36, 37). Though, the hypothesis that DPRs might interact with chaperones was left unexplored.
  • GFP -tagged DPRs were expressed in a non-neuronal human cell line, 293 cells, and it was found that each of the five DPRs lead to the robust induction of HSPA6 and many other disease-associated HSF1 target genes compared to GFP alone. Together, these results suggest that several individual DPRs are sufficient to lead to the transcriptional changes specific to the brains of patients with C90RF72-ALS/FTD
  • the HSF 1 pathway which was found to be induced in C90RF72 patients, is highly conserved from budding yeast to mammals and is an important mediator of the compensatory response to disruptions in proteostasis such as heat shock (30).
  • the gene expression changes found in the C90RF72 brain provided a validated signature that could be used as a probe for determining which of the proposed pathogenic mechanisms for the repeat expansion was most likely acting in patients. While this study cannot rule out a role for C90RF72 haploinsufficiency or RNA- toxicity, these findings that even transient exposure of human neurons to DPRs is sufficient to induce a response highly correlated to the patient brain lends significant credence to the notion that they are a key driver of the disease process.
  • FIG. 14 the following model for neural degeneration in C90RF72-ALSfFTO is proposed (FIG. 14).
  • the presence of the C90RF72 repeat expansions results in the production of various toxic DPRs.
  • neurons can degrade DPRs or perhaps sequester them into protective p62-positive inclusions.
  • With aging there is a decreased capacity of neurons to maintain proteostasis, and environmental insults may be associated with additional proteotoxic stress, leading to the further accumulation of DPRs.
  • Neurons initially compensate through upregulation of an HSFl response and increased expression of the chaperome networks.
  • hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non- ATG translation in c9FTD/ALS. Acta Neuropathol 126, 829-844 (2013).

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Neurosurgery (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Neurology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Psychiatry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne l'identification d'une signature du gène C90RF72 pour la SLA et des méthodes destinées à induire la signature du gène C90RF72 pour la SLA dans les neurones. L'invention concerne des méthodes d'utilisation de la signature du gène C90RF72 pour le dépistage de troubles neurodégénératifs, le diagnostic de troubles neurodégénératifs, et l'identification d'agents pour le traitement de troubles neurodégénératifs.
PCT/US2018/036980 2017-06-09 2018-06-11 Signature génétique et biomarqueurs pour la sla et la démence Ceased WO2018227212A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762517567P 2017-06-09 2017-06-09
US62/517,567 2017-06-09

Publications (1)

Publication Number Publication Date
WO2018227212A1 true WO2018227212A1 (fr) 2018-12-13

Family

ID=64566308

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/036980 Ceased WO2018227212A1 (fr) 2017-06-09 2018-06-11 Signature génétique et biomarqueurs pour la sla et la démence

Country Status (1)

Country Link
WO (1) WO2018227212A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244375A (zh) * 2021-05-07 2021-08-13 浙江大学 一种用于减少聚甘氨酸-丙氨酸聚集和减轻行为缺陷的组合物及其应用
CN116672341A (zh) * 2022-02-23 2023-09-01 中国科学院上海药物研究所 雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110124104A1 (en) * 2008-05-20 2011-05-26 Andrei Gudkov Inducing cell death by inhibiting adaptive heat shock response
WO2016210372A2 (fr) * 2015-06-25 2016-12-29 University Of Southern California Procédés de traitement de maladies neurologiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110124104A1 (en) * 2008-05-20 2011-05-26 Andrei Gudkov Inducing cell death by inhibiting adaptive heat shock response
WO2016210372A2 (fr) * 2015-06-25 2016-12-29 University Of Southern California Procédés de traitement de maladies neurologiques

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Product Data Sheet: Z-Leu-Leu-Leu-B(OH)2 [MG -232]", ADIPOGEN LIFE SCIENCES, 27 October 2014 (2014-10-27), XP055558541, Retrieved from the Internet <URL:https://adipogen.com/productmanagement/resource/download/type/sheet/id/11437> [retrieved on 20180828] *
CHA, J ET AL.: "A Novel Small Molecule HSP90 Inhibitor, NXD30001, Differentially Induces Heat Shock Proteins In Nervous Tissue In Culture And In Vivo", CELL STRESS AND CHAPERONES, vol. 19, no. 3, 3 October 2013 (2013-10-03), pages 421 - 435, XP055558525 *
CHANG, Y ET AL.: "The Glycine-Alanine Dipeptide Repeats From C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell -to- Cell Transmission Properties", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 291, no. 10, 14 January 2016 (2016-01-14), pages 4903 - 4911, XP055524348 *
CHEN, H ET AL.: "The Heat Shock Response Plays An Important Role In TDP-43 Clearance: Evidence For Dysfunction In Amyotrophic Lateral Sclerosis", BRAIN, vol. 139, 1 March 2016 (2016-03-01), pages 1417 - 1432, XP055558581 *
GUPTA, R ET AL.: "The Proline/Arginine Dipeptide From Hexanucleotide Repeat Expanded C90RF72 Inhibits the Proteasome", ENEURO, vol. 4, no. 1, January 2017 (2017-01-01), pages 1 - 18, XP055558521 *
LOPEZ-GONZALEZ, R ET AL.: "Poly (GR) In C90RF72-Related ALS/FTD Compromises Mitochondrial Function And Increases Oxidative Stress And DNA Damage In iPSC-Derived Motor Neurons", NEURON., vol. 92, no. 2, 6 October 2016 (2016-10-06), pages 383 - 391, XP029778109 *
VLEMINCKX, V ET AL.: "Upregulation Of HSP27 In A Transgenic Model Of ALS", JOURNAL OF NEUROPATHY AND EXPERIMENTAL NEUROLOGY, vol. 61, no. 11, November 2002 (2002-11-01), pages 968 - 974, XP055558528 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244375A (zh) * 2021-05-07 2021-08-13 浙江大学 一种用于减少聚甘氨酸-丙氨酸聚集和减轻行为缺陷的组合物及其应用
CN116672341A (zh) * 2022-02-23 2023-09-01 中国科学院上海药物研究所 雷帕霉素在治疗渐冻症和额颞叶痴呆中的应用

Similar Documents

Publication Publication Date Title
Yang et al. Lonicera japonica extends lifespan and healthspan in Caenorhabditis elegans
Wodrich et al. The unfolded protein responses in health, aging, and neurodegeneration: recent advances and future considerations
Gendron et al. Disease mechanisms of C9ORF72 repeat expansions
Nguyen et al. Repeat-associated non-ATG translation: molecular mechanisms and contribution to neurological disease
Mangold et al. Sexually divergent induction of microglial-associated neuroinflammation with hippocampal aging
Gendron et al. Mechanisms of toxicity in C9FTLD/ALS
Seo et al. Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome
Rusmini et al. The role of the protein quality control system in SBMA
Moily et al. Transcriptional profiles for distinct aggregation states of mutant Huntingtin exon 1 protein unmask new Huntington's disease pathways
Sanuki et al. Normal aging hyperactivates innate immunity and reduces the medical efficacy of minocycline in brain injury
EP3034074A1 (fr) Composé pour le traitement de la dystrophie myotonique de type 1
Zhang et al. Long non-coding RNA KCNQ1OT1 promotes hydrogen peroxide-induced lens epithelial cell apoptosis and oxidative stress by regulating miR-223-3p/BCL2L2 axis
Botta-Orfila et al. Molecular pathophysiology of fragile X-associated tremor/ataxia syndrome and perspectives for drug development
WO2018227212A1 (fr) Signature génétique et biomarqueurs pour la sla et la démence
Dong et al. USF2 activates RhoB/ROCK pathway by transcriptional inhibition of miR-206 to promote pyroptosis in septic cardiomyocytes
Butler et al. Murine social stress results in long lasting voiding dysfunction
Tesla et al. Benzoxazole-derivatives enhance progranulin expression and reverse the aberrant lysosomal proteome caused by GRN haploinsufficiency
Chong et al. Pathogenesis underlying hexanucleotide repeat expansions in C9orf72 gene in amyotrophic lateral sclerosis
Jang et al. Recombinant RAGE antagonist peptide promotes alveolar epithelial cell regeneration via the RAGE/MAPKs/MMP2 pathway in emphysema
CN114431193B (zh) Prrc2b在制备治疗脱髓鞘疾病的药物中的应用
Mikuriya et al. Edaravone mitigates TDP-43 mislocalization in human amyotrophic lateral sclerosis neurons with potential implication of the SIRT1-XBP1 pathway
Yu et al. A mechanistic analysis of metformin's biphasic effects on lifespan and healthspan in C. elegans: Elixir in youth, poison in elder
CN119318706A (zh) Nr3a作为蛋白靶点在筛选或制备预防和/或治疗抑郁样行为的药物中的应用
Klementa et al. Acute kidney injury due to gentamicin nephrotoxicity and specific miRNAs as biomarkers.
Yu et al. Dendrobium officinale phenolic extract maintains proteostasis by regulating autophagy in a Caenorhabditis elegans model of Alzheimer's disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18813517

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18813517

Country of ref document: EP

Kind code of ref document: A1