Understanding the impact of Schizophrenia and Autism risk genes on brain development

Karun K. Singh, Ph.D.

Stem Cell and Cancer Research InstituteDepartment of Biochemistry and Biomedical Sciences

McMaster University

Normal Brain Development

`Genes and Neurodevelopment

Genetic Mutation

Brain Development Disorder

SCHIZOPHRENIA

AUTISM

`Symptoms of Schizophrenia• Positive symptoms

– Hallucinations – Delusions– Thought disorder (confused thinking and speech)

• Negative symptoms– Diminished emotional expression– Diminished motivation – Inability to experience pleasure

• Cognitive symptoms– Poor concentration– Difficult to plan and organize– Poor memory

• Treatments– Antipsychotic drugs (old and new

generation)– Cognitive behavior therapy

`Neuropathology of Schizophrenia

• Reduction of brain volume (Grey Matter)• Lateral Ventricle enlargement• Decreased GABAergic interneurons (GABA and GAD67 staining)• Less extensive arborization/neuronal complexity (dendritic/synapse)• OVERALL – Nothing absolutely conclusive from brain imaging

Unaffected twin

N Eng J Med. 1990.

Schizophrenic twin

`Genetics plays a major role in psychiatric disorders

(Tom Insel, Director NIMH, JCI, 2010)

**Complex genetics at play in psychiatric disorders**

2011

Loci for SZ

mir137CSMD1TRIM26PCGEM1

Loci for BPD

ITIH3-ITIH4 region

• Exome Sequencing• Whole Genome Sequencing

…more and more genes!

`Modeling the Genetics of Neurodevelopmental

and Psychiatric Disorders

Brain Disease Risk Gene

Developing Mouse Brain

Human CellularReprogramming

Patient Cells

Why do this?1. Understand how genes impact brain structure/function during development2. Understand core signaling pathways affected

Disrupted in Schizophrenia-1 (DISC1)

Chromosome (1; 11) translocationdisrupts the DISC1 locus.

`

Approach: In utero electroporation to study neural stem cells and neuronal differentiation/maturation

Experimental model to examine DISC1/Dixdc1: The developing mouse cortex

CP - neurons

IZ – axon tracts

VZ – neural stem cells

Dire

ction

of m

igra

tion

Singh et al., 2010

`Dixdc1 functionally interacts with DISC1 to

regulate neural progenitor proliferation

`Dixdc1 and DISC1 regulate neuronal migration

Singh et al., 2010

DISC1 DIXDC1XFrag2

Early brain development

Mid-brain development

`Dixdc1 is a critical regulator of DISC1 and

embryonic brain development

Singh et al., 2010

`Does Dixdc1 functionally interact with DISC1 in

neural connectivity development?

Penzes et al., Nat Neurosci 2011

• Dendrite Growth

• Spine Structure

• Synapse Formation

`Does a DISC1-Dixdc1 pathway regulate the

growth of dendrites and synapses?

10 30 50 70 90110

1300

2

4

6

8

10

12

Sholl Analysis

Ctrl shRNADisc1 shRNADixdc1 shRNADix Frag2

Distance from Soma (μm)

Num

ber o

f Int

erse

ction

s

Ctl shRNA

DISC1 sh

RNA

Dixdcsh

RNA

DixFrag

20

100200300400500600700800900

Total Dendrite Branch Length

Tota

l Bra

nch

Leng

th (µ

m)

Vickie Kwan, preliminary results

Dixdc1 Frag2Control shRNA DISC1 shRNA Dixdc1 shRNA

DIV6

Confirming this in vivo in utero electroporation

`Examining synapses with genetic tools:

Trans-synaptic labeling using Rabies virus

Ed Callaway LabSalk Institute

Advantages

1. Single Cell Resolution

2. Watch dynamics over time

`Trans-synaptic labeling in mouse cortical neuronsIUE E15 Culture E17 DIV 10 DIV 14

Infect:Rabies

Fix cellsLV-Syn-HTG

Rabies virus courtesy of Ian Wickersham and Heather

Sullivan (Seung Lab@MIT)How does DISC/Dixdc1 regulate synapse formation?

`Studying the Genetics of Autism

Aldinger et al., 2011 Neuron

Not included:

• Additional CNVs• Exome sequencing (de novo mutations)• Whole genome

sequencing

`Human cellular reprogramming to create

patient-derived neural cells

Advantages• Model autism genes• Behavior assays• Neural circuits and

cell types involved

Some Disadvantages• Difficult to model disease

gene networks, large CNVs, human SNPs

• Gene disruption doesn’t completely mimic human genetics (eg. KO≠mutation)

• Not patient brain tissue (human brain specific)

`Human cellular reprogramming to create

patient-derived neural cells

patient skin samples

Reprogramming

genesDifferentiate

Induced pluripotent stem (iPS) cells

Patient-derivedNEURAL CELLS

Induced Neuronal (iN) or Neural Progenitor (iNP) cells

Direct conversion

ADVANTAGES of Direct Conversion:1. Faster than iPS method2. Epigenetic signature of patient cell is likely preserved3. Specific Neuronal subtype generation (Spinal Motor, Dopaminergic)

`Studying the Genetics of Autism

Aldinger et al., 2011 Neuron

`Tuberous Sclerosis Complex (TSC): a genetic

disorder with high rates of autismCollaboration with Dr. Philippe Major, Sainte-Justine Hospital, Montreal1. Benign tumors in vital organs including brain2. Autism features (syndromic autism) 25-60% in ASD3. Learning disabilities, developmental delay4. Epilepsy

Kelleher, III and Bear, 2012 CELL

Mouse Models• Protein

Translation

• Plasticity (mGluR-LTD)

`Using Cellular Reprogramming to Study

TSC and Autism

Healthy or TSC

Fibroblasts

HumanNeuralCells

Direct

Cellular

Repro

gramming

DrugScreen

Phenotyp

e and

Assay

Dev

elopmen

t

Synapse FunctionDendrite/Spine GrowthTrans-synaptic labelingAutomated Electrophysiology

`In vivo Human Neuronal Model

using Xenotransplantation

Fluorescent Label

Patient-derived neural cells

Transplant

In vivo profile of human cells:

• How do patient neural cells functionally integrate into the developingor adult brain?

Acknowledgements

My lab:Vickie KwanShashwat DesaiOmar Shehab

University of Montreal-Sainte Justine Hospital

Dr. Philippe Major (TSC)

Funding:

Ontario Research Fund

Massachusetts Institute of Technology:Dr. Li-Huei Tsai