Autism Spectrum Disorders; heterogeneous nature of genetic and brain pathology in ASD– which makes it difficult to produce relevant animal and cell models

1. E R I N M I L L E R
P A T R I C K S A V O I E
M A S U M A A K T E R
Altered proliferation and networks in
neural cells derived from idiopathic
autistic individuals

2. Autism Spectrum Disorders (ASD)
 ASDs are a group of complex neurodevelopmental disorders
 ASDs are characterized by impaired social interaction and
limited/repetitive interests and behaviors
 About 1 in 59 children have been identified with ASD
 ASDs are phenotypically and etiologically heterogeneous
 Challenging to study due to heterogeneous nature of genetic and brain
pathology in ASD– which makes it difficult to produce relevant animal
and cell models
 Non-syndromic (idiopathic) form of ASD is not clearly linked to other
neurological diseases; this is the majority of ASD
 Syndromic (secondary) ASD has a clear cause is recognized

3. Pathophysiology of ASD
 Imaging and gene expression studies of post-mortem brains from ASD
patients revealed disruption of developmental and proliferation gene
networks
 Gene expression changes in post-mortem ASD brains overlap with
developmentally regulated genes (Cell-cycle, cortical patterns,
proliferation, neural differentiation)
 A certain subset of individuals with ASD display macrencephaly
(~20%-30%) and altered brain growth trajectory
 In some ASD cases, there is an abnormal increase in brain size in
during the first 3 years of life with excess number of neurons that
precedes the first clinical signs

4. Focus of Study
 Researchers reasoned that patients with ASD sharing early brain
enlargement varying from mild to extreme may also share underlying
cellular pathway dysregulation
 Wanted to use iPSCs to model idiopathic ASD and reveal possible
cellular and molecular mechanisms that could underlie brain
abnormalities
 Possibly leading to better understanding, ASD biomarkers, and targets
for therapy
 Reprogrammed fibroblasts to generate iPSCs, NPCs, & neurons from
ASD patients with brain overgrowth early in life to look for common
cellular & molecular pathway dysregulation

5. Patient Ascertainment
 Subjects were selected from lists of people that had been identified and
diagnosed with ASD and had MRI scans as toddlers
 From among these subjects, they selected those with larger than normal
average brain volume as compared to typically developing toddlers and
also who demonstrated behavioral criteria consistent with autism as
defined by the DSM-IV
 Control group selected randomly from lists of typically developing
individuals who had an MRI as toddlers and had no history of
psychological, genetic, or other disorders

6. Methods
 Copy Number Variation Analyses
Comparison of CNVs in a control group with those of ASD
patients.
 Exome Sequencing
Involves sequencing of all protein coding regions of genes in a
genome. Researchers cultured fibroblasts from 8 ASD patients
then extracted DNA.

7. iPSCs & Cellular Reprogramming
 Reprogramming of somatic cells to a pluripotent
state through over-expression of specific genes
 To accomplish this, they transduced the fibroblasts
with retroviruses containing OCT4, SOX2, KFL4,
and MYC to induce overexpression of these genes
 Performed experiments with at least 2 independent
iPSCs clones to avoid potential variability caused by
retroviral insertions

8. Methods
 Immunofluorescence
Use of antibodies that are labeled with fluorescent dyes, which
help to provide greater contrast when viewing under a
microscope. The labeled antibodies bind to antigens on the
sample being viewed.
 Western Blotting
Used to detect the presence of proteins that are possibly specific
to or more prominent in patients with an ASD. Provides a
visualization of the proteins that are present.
 Neural Differentiation Analysis
Researches cultured iPSC colonies then transferred them to an
NPC medium. The NPCs were then induced to form neurons.

9. Methods
 Whole Cell Patch Clamp + Electrophysiology
Analysis
Used to study ionic currents in cells, specifically used to learn
more about the electrophysiology of ASD cells. Comparing them
with a control group.
 RNA Sequencing
Researchers compared the RNA of ASD individuals to that of a
control group.

10. Question: How alteration of the NPC proliferation rate contribute to brain size?
Method: Quantitative MRI-validated early brain enlargement Cell culture, cell cycle analysis
ASD Individuals with macrencephaly proliferated faster and proliferation contributes to the large brain early on in ASD
The ASD donors displayed larger brain size compared to the
normal average brain size
➢ From P4, the population doubling time decreased in ASD NPCs
compared to control NPCs.
➢ The shortening of G1 and S phases is the main reason for the
decrease in the population doubling time but no change of G2-M
phase length
Ki67+=
cycling
cell
pHH3+Ki67
-G2-M
phase
mitotic cells
➢ An increased percentage of Ki67+ (cycling cells) in ASD relative to
control NPCs
➢ Percentage of pHH3+Ki67+ (G2-M phase mitotic cells) was unaffected in
autistic NPCs
Positive correlation with NPC proliferation and an inverse
correlation with NPC doubling time

11. Question: How alterations in the canonical Wnt pathway govern the aberrant proliferation in ASD-derived NPCs?
Methods: Genomic analyses ,TOP-flash assays, immunocytochemistry, Western blot analysis
Providing that reduced β-catenin transcriptional activity and transcription factor Brn2 effect cell proliferation of autistic NPCs
LiCl-an inhibitor of GSK3, to activate
Wnt signaling
Wnt3A, one of Wnt family members
➢ β-catenin transcriptional activity was indeed reduced in ASD NPCs
➢ Activity was significantly reduced in LiCl-treated ASD NPCs compared to
control NPCs, suggesting that the cause of reduced β-catenin
transcriptional activity is downstream of GSK3β
➢ moderate increase of Wnt is sufficient to rescue abnormal over
proliferation in ASD by increasing doubling time
➢ BRN2 plays a key role in regulating cellular proliferation and regulate
neurogenesis
➢ The percentage of BRN2+ cells was in fact reduced in ASD NPCs compared to
controls
➢ BRN2 protein levels were reduced in ASD NPCs compared to controls

12. ➢ The number of proliferation cells in ASD NPCs was similar to
controls after BRN2 overexpression
➢ The expression pattern of BRN2 and Ki67 in the BRN2 transfected
ASD NPCs resembled that of control NPCs
Question: whether exogenous BRN2 rescued the increased rate of proliferation in ASD NPCs?
Methods: transfected CAG-BRN2, immunocytochemistry, Western blot analysis
Excitatory cell fate determination NGN2+
Inhibitory cell fate determination (MASH1, DLX2 and
NKX2.1)
➢ Reduction in the percentage of glutamatergic NGN2+
NPCs in ASD NPCs
➢ GABAergic inhibitory precursors were up-regulated in
ASD
Question: whether glutamatergic and GABAergic cell fate determination were affected in ASD NPCs?
Fate determination didn’t have alteration in ASD neuronal survival after differentiation

13. Question: How alteration of neuronal differentiation happen in ASD NPCs?
Methods: immunocytochemistry
significant reduction in inhibitory
excitatory neurotransmitters
GABA in ASD neurons
VGLUT1 (vesicular
glutamate transpoter-1)
PSD95 (postsynaptic
density-95)
significant reduction in the density of both Synapsin and VGLUT1 (vesicular glutamate
transpoter-1) puncta from ASD neurons

14. Question: Whether the decreased excitatory synapse affect the expression of neurotransmitters of ASD-derived neurons or
not?
Methods: qPCR array of 84 neurotransmitter receptors-related genes
➢ Down-regulated GABA-related and neuropeptide Y receptors in ASD derived neurons
➢ Down-regulation of the synapse-related genes, such as KCNA1, TH, PHOX2A and DRD3, in ASD neurons
➢ Genes related to early stages of neural differentiation, such as NEUROG3, FOXG1, SOX10 and P2RY2, were significantly
upregulated in ASD.

15. Question: whether the functional properties of iPSC-derived neurons differ in ASD individual compare to control or not?
Methods: Electrophysiological recordings by multielectrode arrays (MEA)
Insulin-like growth factor-1 (IGF-1)
function in both brain development an
plasticity
➢ The number of spikes increased in control neurons compared to ASD did not, concluding a significant
difference between ASD and control networks
➢ Reduced number of synchronized bursts in ASD individual
➢ ASD patients displayed a tendency to improve neuronal spike number when IGF-1 were treated
MEA Contain multiple electrodes essentially serving as neural interfaces that connect neurons to electronic circuitry.
Network bursting is the presence of an
excitatory population of oscillatory
neurons

16. Question: Gene expression pattern changes at the NPC and neuronal stages in ASD or not?
Methods: Differential expression (DE) analysis, GO enrichment analysis, Weighted Gene Co-expression Network Analysis
(WGCNA)
71 genes that were significantly differentially expressed in ASD compared to controls at the
NPC stage and 154 genes at the neuronal stage, which is clearly distinguished by hierarchical
clustering
ASD CLT CLT ASD
The expression fold changes of the top
dysregulated genes
The expression fold changes of the top
dysregulated genes
Down Up Down Up

17. Visualization of striking clustering pattern of the case-associated genes and known ASD susceptibility genes (curated from SFARI database)
Question: Gene expression pattern changes at the NPC and neuronal stages in ASD or not?
Methods: Differential expression (DE) analysis, GO enrichment analysis, Weighted Gene Co-expression Network Analysis
(WGCNA)

18. Question: whether transcriptional alterations associated with early brain overgrowth in ASD or not?
Identified 35 genes showing significant differences in
their expression trajectories between ASD NPCs and
controls in the progenitor to neuron transition
➢ significantly enriched for voltage-gated cation channels e.g. CACNG5,
KCNA6, KCNC2, and KCNIP2
➢ Those channel genes were up-regulated in control cells, but this increase
was attenuated in the ASD cells
Voltage-gated cation channel dysregulation in ASD neural differentiation, consistent with decreased excitatory glutamatergic
synapses, likely affecting synaptic transmission.

19. Discussion:

20. Summary of Significant Findings
 Researchers hypothesized that increased brain
volume and neurons found in ASD may result from
increased rates of proliferation of NPCs
 NPCs derived from iPSCs from ASD patients did
display rapid rates of proliferation and showed
abnormal differentiation compared to controls
 Abnormalities in proliferation of NPCs can lead to
long lasting differentiation abnormalities, such as
ASD
 Uncovered more information about possible
mechanisms for IGF-1 treatment

21. Future Research
 Unclear if proliferation and differentiation defects
result from macrencephaly, ASD, or both
 Future studies needed to distinguish this
 Using normocephalic ASD-patient derived NPCs and
neurons
 iPSC models of macrencephaly in those without ASD