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Neurodevelopmental Disorders
Focus: Rare Diseases
Margot Mayer-Proschel, John Foxe, Jonathan Mink,
Mark Noble, Alex Paciorkowski, Chris Proschel
Alex Paciorkowski
Chris Proschel
Jonathan Mink
Margot
Mayer-Proschel
Mark Noble
FOXG1
Disorders
MECP2
disorders
Developmental
Brain
Disorders
Fragile-
X
Vanishing White Matter
16p11.2
Ataxia Telangiectasia
MLD Gaucher
Krabbe BattenDisease
Cystinosis
NPC
Rett
Syndrome
• Drug discovery through identification of common
denominators• Broad impact
John Foxe
Congenital diseases
Variable disease onset
Secondary insults
modulate disease
Misfolded proteins
ER stress
Mitochondrial defects
Inflammation
Oxidative stress
A Common Denominator
Lysosomal dysfunction Drugs
Congenital diseases
Variable disease onset
Secondary insults
modulate disease
Genetic predisposition
Variable disease onset
Secondary insults
contribute to disease
Misfolded proteins
ER stress
Mitochondrial defects
Inflammation
Oxidative stress
A Broader Impact
Lysosomal dysfunction Drugs
Cellular
endpoints
• Proliferation
• Differentiation
• Survival
• Neurite outgrowth
Drug screen
• FDA approved drug screen
• Confirm candidate “hits”
• Verification in relevant human
iPSC cells
Structural Analysis
• Identify BBB permeable drug
• Test in animal model
• Test for off target effects
Clinical application
• Identification of clinical trial
cohorts
• Analysis of carriers
• Relevant “recovery” readouts
Genetic
• Disease phenotype
• Clinical history
• Identification of new mutations
• Cohort access
General Concept
Oligodendrocyte
Progenitors
• Proliferation
• Differentiation
• Lysosomal
Function
Drug Screen
• Identify candidates
• Confirm cellular rescue
• Effect on lysosome
• Test outcome in iPSCs
• Identify harmful drugs.
Structural Analysis
• Identified BBB permeable drugs
• Test in animal model (Twi)
• No reported CNS toxicity
Clinical application
• Identification of clinical trial
cohorts
• Clinical history of patients
receiving the “bad drug”
• Analysis of carriers?
Krabbe Disease
• Lysosomal storage disease
• Known mutation in GALC
• Heterozygosity associated
with mild phenotype
A Specific Example
P10 P40P25P20P15 P35P30
symptomonset
twitchingweight loss /
muscle wastingdeath
twi/twi P5P0
Brain Psydetected
Postnatal age (days)
Krabbe Disease:
• Severe, progressive loss of myelin & neurodegeneration
• Enzymatic deficiencies (mutations) in galactocerebrosidase (GALC)
• Accumulation of the toxic lipid Psychosine (Psy)
• Twitcher mouse: a pathologically/genetically authentic murine model
WT
twi
DAPI fluoromyelin
P10 P40P25P20P15 P35P30
symptomonset
twitchingweight loss /
muscle wastingdeath
twi/twi P5P0
Brain Psydetected
Postnatal age (days)
Krabbe Disease:
• Severe, progressive loss of myelin & neurodegeneration
• Enzymatic deficiencies (mutations) in galactocerebrosidase (GALC)
• Accumulation of the toxic lipid Psychosine (Psy)
• Twitcher mouse: a pathologically/genetically authentic murine model
WT
twi
DAPI fluoromyelin
*
WT twi
P40 OLs
**
P40 OPCs
WT twi
*
WT twi
P15 OPCs
*
WT twi
P40 OLs
**
P40 OPCs
WT twi
Oligodendrocyte
Progenitors
• Proliferation
• Differentiation
(Lysosomal
Function)
Drug Screen
• 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
Clinical application
• Identification of clinical trial
cohorts
• Clinical history of patients
receiving the “bad drug”
• Analysis of carriers?
Krabbe Disease
• Lysosomal storage disease
• Known mutation in GALC
• Heterozygosity associated
with mild phenotype
A Specific Example….
…and also disrupts endocytosis, lipid metabolism, and the
activity of resident enzymes.
†
PND 17
WT twi
OPC pH
*
Isolated progenitor cells recapitulate in vivo defects
Proliferation Lysosomal pH
Oligodendrocyte
Progenitors
• Proliferation
• Differentiation
(Lysosomal
Function)
Drug Screen
• 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
Clinical application
• Identification of clinical trial
cohorts
• Clinical history of patients
receiving the “bad drug”
• Analysis of carriers?
Krabbe Disease
• Lysosomal storage disease
• Known mutation in GALC
• Heterozygosity associated
with mild phenotype
A Specific Example….
Focus on correcting important cellular behaviors, not on specific proteins or genes
Unbiased screening
Psy1052
Drugs
Cell division
Validation of
‘hits’
Restoration
of lysosomal
function?
calcein-AM (live) PI (dead)
+ vehicle (DMSO) + psychosine0h 24h 48h 72h
342 632 1630 3192
Elapsed time:
Cell count:
+ psy
1104
++++
1303
1305
1107
c c c
b
+++++++
808
1002
704
611
907
906
++++++208
211
105
210
211
c c cc
c cc
aa b
aba
+
610
+ veh
+ Psy
Reduce Psy toxicity
2400 drugs@ 3 concentrations
: + Psy
Enhance Psy toxicity?
psy
NK
Hclo
fIG
F-1
+++ +
**
a b a
CathD
activity
†
ba a
+ psy
NK
H
+
IGF-1
+
clo
f
+
Endocytosis Lipid accumulation
clo
fN
KH
IGF-1
+ + + + psy
b
psy
910
+
906
+
505
+
104
+
NT-3
+
IGF-1
+
NK
H
+
clo
f
++
** *** * * *
a a a
Lysosomal pH
Top hits rescue cellular defect associated with lysosomal
dysfunction in vitro
Oligodendrocyte
Progenitors
• Proliferation
• Differentiation
(Lysosomal
Function)
Drug Screen
• 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
Clinical application
• Identification of clinical trial
cohorts
• Clinical history of patients
receiving the “bad drug”
• Analysis of carriers?
Krabbe Disease
• Lysosomal storage disease
• Known mutation in GALC
• Heterozygosity associated
with mild phenotype
A Specific Example….
Top hits rescue cellular defect associated with
lysosomal dysfunction in vivo
(A) Stance time (B) Break time (C) Propel time
c
††
a
†
c
WT twi WT twi WT twi
Weight gain
*!
BMT
AAV
veh
NK
H
twi
Survival
*
NK
H
ve
h
WT
ve
h
twi
WT
ve
hT
wi
ve
hT
wi
NK
H
DAPI fluoromyelin
*
NK
H
ve
h
WT
ve
h
twi
(A) PND40 Myelin (B) PND40 Myelin (C) PND40 Oligodendrocytes
Myelination
(A) Stance time (B) Break time (C) Propel time
c
††
a
†
c
WT twi WT twi WT twi
Weight gain
*!
BMT
AAV
veh
NK
H
twi
Survival
*
NK
H
ve
h
WTv
eh
twi
WT
ve
hT
wi
veh
Tw
i N
KH
DAPI fluoromyelin
*
NK
H
ve
h
WT
ve
h
twi
(A) PND40 Myelin (B) PND40 Myelin (C) PND40 Oligodendrocytes
Survival Motor function (A) Stance time (B) Break time (C) Propel time
c
††
a
†
c
WT twi WT twi WT twi
Weight gain
*!
BMT
AAV
veh
NK
H
twi
Survival
*
NK
H
ve
h
WT
ve
h
twi
WT
ve
hT
wi
ve
hT
wi
NK
H
DAPI fluoromyelin
*
NK
H
ve
h
WT
ve
h
twi
(A) PND40 Myelin (B) PND40 Myelin (C) PND40 Oligodendrocytes
Oligodendrocyte
Progenitors
• Proliferation
• Differentiation
(Lysosomal
Function)
Drug Screen
• 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
Clinical application
• Relevant “recovery” readouts
Physiology
Imaging
• Identification of clinical trial
cohorts
• Clinical history of patients
receiving the “bad drug”
• Analysis of carriers?
Krabbe Disease
• Lysosomal storage disease
• Known mutation in GALC
• Heterozygosity associated
with mild phenotype
A Specific Example….
Glaucoma
MS
Krabbe
disease
Jonathan, Alex, John
Chris Margot
Mark
Define cellular
endpoints
Establish cell
system
Test candidate
lysosomal drugs on
target cells
Patient cohorts• Access
• Genetics, Disease
Specific Natural History
Krabbe, Rett, Gaucher,
Hurler, Batten, MLD, A-T,
VWM
Clinical trial design• Relevant “recovery”
readouts
• Diagnostic/ Biomarkers
Test in iPSCs with distinct
genetic background
Proposed time line and human capital
Confirm candidate “hits”
using in vivo models
6-12 months 12 months12 months
Human
iPSCs
Mouse
mutant
2 PhD
2 TAs
MDs
1 PhD
Statisticia
n
2 PhD
3 TAs
MDs
1 PhD
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):6331-
49.
• 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
2. Broad Impact
Drug discovery