neurodevelopmental disorders focus: rare diseases · neurodevelopmental disorders focus: rare...

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


Secondary insults

modulate disease

Genetic predisposition


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



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






Structural Analysis/In vivo






cohorts




Krabbe Disease




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












cohorts




Krabbe Disease




with mild phenotype


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












cohorts




Krabbe Disease




with mild phenotype


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


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


Survival Motor function (A) Stance time (B) Break time (C) Propel time

c

††

a

†

c


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


Oligodendrocyte

Progenitors

• Proliferation

• Differentiation

(Lysosomal

Function)

Drug Screen











• Relevant “recovery” readouts

Physiology

Imaging


cohorts




Krabbe Disease




with mild phenotype


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

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