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Neurodevelopmental Disorders Focus: Rare Diseases Margot Mayer-Proschel, John Foxe, Jonathan Mink, Mark Noble, Alex Paciorkowski, Chris Proschel Mark Noble Alex Paciorkowski MLD Gaucher Jonathan Mink MECP2 disorders FOXG1 Disorders Developmental Brain Disorders Krabbe • Drug discovery through identification of common denominators • Broad impact FragileX Vanishing White Matter Chris Proschel Ataxia Telangiectasia Batten Disease Rett Syndrome Cystinosis NPC John Foxe 16p11.2 Margot Mayer-Proschel A Common Denominator Oxidative stress Mitochondrial defects Congenital diseases Variable disease onset ER stress Misfolded proteins Secondary insults modulate disease Inflammation Lysosomal dysfunction Drugs A Broader Impact Oxidative stress Mitochondrial defects Congenital diseases Variable disease onset ER stress Misfolded proteins Secondary insults modulate disease Genetic predisposition Variable disease onset Secondary insults Inflammation Lysosomal dysfunction contribute to disease Drugs General Concept Cellular endpoints • • • • Proliferation Differentiation Survival Neurite outgrowth Genetic • • • • Disease phenotype Clinical history Identification of new mutations Cohort access Drug screen • FDA approved drug screen • Confirm candidate “hits” • Verification in relevant human iPSC cells Clinical application • Identification of clinical trial cohorts • Analysis of carriers • Relevant “recovery” readouts Structural Analysis • Identify BBB permeable drug • Test in animal model • Test for off target effects A Specific Example Oligodendrocyte Progenitors • Proliferation • Differentiation • Lysosomal Function Krabbe Disease • Lysosomal storage disease • Known mutation in GALC • Heterozygosity associated with mild phenotype Drug Screen Clinical application • Identify candidates • Confirm cellular rescue • Effect on lysosome • Test outcome in iPSCs • Identify harmful drugs. • Identification of clinical trial cohorts • Clinical history of patients receiving the “bad drug” • Analysis of carriers? Structural Analysis • Identified BBB permeable drugs • Test in animal model (Twi) • No reported CNS toxicity Accumulation of the toxic lipid Psychosine (Psy) Twitcher mouse: a pathologically/genetically authentic murine model Brain Psy detected symptom onset P5 * P10 P15 P40 OP P40 OPCs twitching * P20* P25 weight loss / muscle wasting P30 P35 ** ** Postnatal age (days) WT twi DAPI WTfluoromyelin twi WT twi WT P0 P40 OLs P40 OLs WT twi/twi P15 OPCs twi twi Brain Psy Disease: symptom weight loss / Krabbe twitching death detected onset muscle wasting P0 • Severe, P5 progressive P10 P15 P30 P35 P40 loss of P20 myelin &P25 neurodegeneration • Enzymatic deficiencies (mutations) in galactocerebrosidase (GALC) • Accumulation of the toxic lipid Psychosine (Psy) • Twitcher mouse: a pathologically/genetically authentic murine model Postnatal age (days) WT twi WT twi A Specific Example…. Oligodendrocyte Progenitors • Proliferation • Differentiation (Lysosomal Function) Drug Screen Krabbe Disease • Lysosomal storage disease • Known mutation in GALC • Heterozygosity associated with mild phenotype Clinical application • Identification of clinical trial cohorts • Clinical history of patients receiving the “bad drug” • Analysis of carriers? • Identify candidates • Confirm cellular rescue • Effect on lysosome • Test outcome in iPSCs • Identify harmful drugs. Structural Analysis/In vivo • Identified BBB permeable drugs • Test in animal model (Twi) • No reported CNS toxicity Isolated progenitor cells recapitulate in vivo defects Proliferation Lysosomal OPC pHpH † * WT twi PND 17 A Specific Example…. Oligodendrocyte Progenitors • Proliferation • Differentiation (Lysosomal Function) Drug Screen Krabbe Disease • Lysosomal storage disease • Known mutation in GALC • Heterozygosity associated with mild phenotype Clinical application • Identification of clinical trial cohorts • Clinical history of patients receiving the “bad drug” • Analysis of carriers? • Identify candidates • Confirm cellular rescue • Effect on lysosome • Test outcome in iPSCs • Identify harmful drugs. Structural Analysis/In vivo • Identified BBB permeable drugs • Test in animal model (Twi) • No reported CNS toxicity Focus on correcting important cellular behaviors, not on specific proteins or genes + veh Psy 1052 Drugs + Psy Restoration of lysosomal function? Validation of ‘hits’ Cell division 0h 24h 2400 drugs 48h @ 3 concentrations 72h : Lysosomal pH 342 ** 632 ** a a a 1630 * * * * + Psy Endocytosis 3192 + vehicle (DMSO) CathD activity + psychosine Lipid accumulation ** † b calcein-AM (live) PI (dead) aa b aba + + +c + psyc c c + + + + psy c a ab cc cc a ab c IGF-1 clof NKH + + + + psy IGF-1 clof NKH + + + + + + + + + psy IGF-1 clof NKH unt: Reduce Psy toxicity Unbiased screening clof NKH IGF-1 NT-3 104 505 906 910 sed me: Enhance Psy toxicity? b Top hits rescue cellular defect associated with lysosomal dysfunction in vitro + + + + + + + + + + + + + + + + + + + psy A Specific Example…. Oligodendrocyte Progenitors • Proliferation • Differentiation (Lysosomal Function) Drug Screen Krabbe Disease • Lysosomal storage disease • Known mutation in GALC • Heterozygosity associated with mild phenotype Clinical application • Identification of clinical trial cohorts • Clinical history of patients receiving the “bad drug” • Analysis of carriers? • Identify candidates • Confirm cellular rescue • Effect on lysosome • Test outcome in iPSCs • Identify harmful drugs. Structural Analysis/In vivo • Identified BBB permeable drugs • Test in animal model (Twi) • No reported CNS toxicity WT twi Top hits rescue cellular defect associated withWT DAPI fluoromyelin WT twi lysosomal dysfunction in vivo (A) Stance time Motor (B) Break time function (A) Stance time AAV † † BMT veh veh NKH NKH c a c WTWTtwitwi c WTWTtwitwi (B) PND40 Myelin Myelination WT veh (C) PND40 Oligodendrocytes Twi veh * fluoromyelin WT twi WT twi NKH veh NKH veh DAPI veh * Twi NKH *! a c WTWTtwitwi (A) PND40 Myelin † † BMT twitwigain Weight (C) Propel time † † AAV (B) Break time twi (C) Propel time veh Survival Survival Survival NKH veh veh WT twi NKH fluoromyelin veh veh T Twi NK DAPI A Specific Example…. Oligodendrocyte Progenitors • Proliferation • Differentiation (Lysosomal Function) Drug Screen • Identify candidates • Confirm cellular rescue • Effect on lysosome • Test outcome in iPSCs • Identify harmful drugs. Krabbe disease Glaucoma MS Structural Analysis/In vivo • Identified BBB permeable drugs • Test in animal model (Twi) • No reported CNS toxicity Krabbe Disease • Lysosomal storage disease • Known mutation in GALC • Heterozygosity associated with mild phenotype Clinical application • Relevant “recovery” readouts Physiology Imaging • Identification of clinical trial cohorts • Clinical history of patients receiving the “bad drug” • Analysis of carriers? Proposed time line and human capital 6-12 months Patient cohorts • Access • Genetics, Disease Specific Natural History Krabbe, Rett, Gaucher, Hurler, Batten, MLD, A-T, VWM Define cellular endpoints 12 months MDs 1 PhD Statisticia n 12 months 2 PhD 2 TAs Clinical trial design Jonathan, Alex, John • Relevant “recovery” readouts • Diagnostic/ Biomarkers Mark Establish cell system Test candidate lysosomal drugs on target cells 2 PhD 3 TAs Confirm candidate “hits” using in vivo models Test in iPSCs with distinct genetic background Mouse mutant Human iPSCs Chris MDs 1 PhD Margot Challenges • • • • Specific cellular targets might not been known (aka ASD) Lysosomal defects might not be a general cellular pathology Patient material might not be readily available for iPSC cell derivation Genetic profile might not be sufficient to identify relevant pathways Solutions • An unbiased approach might identify novel targets (see A-T work) • In addition to lysosomal defects we can screen cells for mitochondrial and ER defects • Crisper/Cas technology might allow to introduce defects in normal cells overcoming the need for patient material • Proteomic or/and lipidomic analysis can be added to the genetic profiling Collaborative published work: • Lysosomal Re-acidification Prevents Lysophingolipid-induced lysosomal impairment and cellular toxicity. Folts C, Scott-Hewitt N, Pröschel C, Mayer-Pröschel M, Noble M. PLOS Biology. 2016 • Epilepsy causing sequence variations in SIK1 disrupt synaptic activity response gene expression and affect neuronal morphology. Pröschel C, Hansen JN, Adil A, Tuttle E, Michelle Lacagnina M, Georgia Buscaglia G, and Marc Halterman M, Paciorkowski A. Eur J Human Genetics, in press • Mutation of ataxia-telangiectasia mutated is associated with dysfunctional glutathione homeostasis in cerebellar astroglia. Campbell A, Bushman J, Munger J, Noble M, Pröschel C, Mayer-Pröschel M. Glia. 2016 Feb;64(2):227-39. • A novel mouse model for ataxia-telangiectasia with a N-terminal mutation displays a behavioral defect and a low incidence of lymphoma but no increased oxidative burden. Campbell A, Krupp B, Bushman J, Noble M, Pröschel C, Mayer-Pröschel M. Hum Mol Genet. 2015 Nov 15;24(22):633149. • EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy. Dietrich J, Lacagnina M, Gass D, Richfield E, Mayer-Pröschel M, Noble M, Torres C, Pröschel C. Nat Med. 2005 Mar;11(3):277-83 A Value-Added Rare Disease Center 1. Common denominator Drug discovery 2. Broad Impact
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