Understanding the impact of Schizophrenia
and Autism risk genes on brain development
Karun K. Singh, Ph.D.
Stem Cell and Cancer Research Institute
Department of Biochemistry and Biomedical Sciences
McMaster University
`
Genes and Neurodevelopment
Normal Brain Development
Brain Development Disorder
SCHIZOPHRENIA
Genetic Mutation
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
N Eng J Med. 1990.
Unaffected twin
•
•
•
•
Schizophrenic twin
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
Genetics plays a` major role in
psychiatric disorders
**Complex genetics at play in psychiatric disorders**
(Tom Insel, Director NIMH, JCI, 2010)
2011
Loci for SZ
• Exome Sequencing
• Whole Genome Sequencing
…more and more genes!
mir137
CSMD1
TRIM26
PCGEM1
Loci for BPD
ITIH3-ITIH4
region
Modeling the Genetics of
Neurodevelopmental
`
and Psychiatric Disorders
Brain Disease
Risk Gene
Developing
Mouse Brain
Patient Cells
Human Cellular
Reprogramming
Why do this?
1. Understand how genes impact brain structure/function during development
2. Understand core signaling pathways affected
Disrupted in Schizophrenia-1 (DISC1)
Chromosome (1; 11) translocation
disrupts the DISC1 locus.
Experimental model to` examine DISC1/Dixdc1:
The developing mouse cortex
Direction of migration
Approach: In utero electroporation to study neural stem
cells and neuronal differentiation/maturation
CP - neurons
IZ – axon tracts
VZ – neural stem cells
Dixdc1 functionally interacts
with DISC1 to
`
regulate neural progenitor proliferation
Singh et al., 2010
`
Dixdc1 and DISC1 regulate
neuronal migration
DISC1 Frag2
X DIXDC1
Singh et al., 2010
Dixdc1 is a critical regulator
of DISC1 and
`
embryonic brain development
Early brain development
Mid-brain development
Singh et al., 2010
Does Dixdc1 functionally
interact with DISC1 in
`
neural connectivity development?
• Dendrite Growth
• Spine Structure
• Synapse Formation
Penzes et al., Nat Neurosci 2011
Does a DISC1-Dixdc1 `pathway regulate the
growth of dendrites and synapses?
Control shRNA
DISC1 shRNA
Dixdc1 shRNA
Dixdc1 Frag2
DIV6
1000
900
800
700
600
500
400
300
200
100
0
14
12
10
8
Ctrl shRNA
6
Disc1 shRNA
4
Dixdc1 shRNA
2
Dix Frag2
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
Total Branch Length (µm)
Total Dendrite Branch Length
Number of Intersections
Sholl Analysis
Distance from Soma (μm)
Confirming this in vivo  in utero electroporation
Vickie Kwan, preliminary results
Examining synapses with genetic tools:
`
Trans-synaptic labeling using Rabies virus
Advantages
1. Single Cell
Resolution
2. Watch dynamics
over time
Ed Callaway Lab
Salk Institute
Trans-synaptic labeling in` mouse cortical neurons
IUE E15
LV-Syn-HTG
Culture E17
DIV 10
Infect:
Rabies
DIV 14
Fix cells
How does DISC/Dixdc1 regulate synapse formation?
Rabies virus courtesy of Ian
Wickersham and Heather
Sullivan (Seung Lab@MIT)
`
Studying the Genetics
of Autism
Not included:
• Additional CNVs
• Exome sequencing
(de novo mutations)
• Whole genome
sequencing
Aldinger et al., 2011 Neuron
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
Induced pluripotent
stem (iPS) cells
Direct conversion
patient skin
samples
Induced Neuronal (iN) or
Neural Progenitor (iNP) cells
Patient-derived
NEURAL CELLS
ADVANTAGES of Direct Conversion:
1. Faster than iPS method
2. Epigenetic signature of patient cell is likely preserved
3. 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 autism
Collaboration with Dr. Philippe Major, Sainte-Justine Hospital, Montreal
1. Benign tumors in vital organs including brain
2. Autism features (syndromic autism) 25-60% in ASD
3. Learning disabilities, developmental delay
4. Epilepsy
Mouse Models
• Protein
Translation
• Plasticity
(mGluR-LTD)
Kelleher, III and Bear, 2012 CELL
Using Cellular Reprogramming to Study
`
TSC and Autism
Healthy or
TSC
Fibroblasts
Human
Neural
Cells
Synapse Function
Dendrite/Spine Growth
Trans-synaptic labeling
Automated Electrophysiology
Drug
Screen
In vivo Human Neuronal
Model
`
using Xenotransplantation
Patient-derived
neural cells
Fluorescent Label
Transplant
In vivo profile of human cells:
• How do patient neural cells functionally integrate into the developing
or adult brain?
Acknowledgements
My lab:
Vickie Kwan
Shashwat Desai
Omar Shehab
Massachusetts Institute
of Technology:
Dr. Li-Huei Tsai
University of MontrealSainte Justine Hospital
Dr. Philippe Major (TSC)
Funding:
Ontario Research Fund