understanding the impact of schizophrenia and autism risk genes on brain development karun k. singh,...
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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
`
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
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)
`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
Cel
lula
r
Repro
gram
min
g
DrugScreen
Phenoty
pe an
d
Assay
Dev
elopm
ent
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?