RESEARCH ARTICLE | NEUROSCIENCE OPEN ACCESS

Neuron-specific transcriptomic signatures indicate

neuroinflammation and altered neuronal activity in ASD
temporal cortex
Pan Zhanga,b,c, Alicja Omanskad,e , Bradley P. Andere,f, Michael J. Gandalc,g,h,i,1,2 , Boryana Stamovae,f,1,2 , and Cynthia M. Schumannd,e,1,2

Edited by Huda Akil, University of Michigan-Ann Arbor, Ann Arbor, MI; received July 12, 2022; accepted December 28, 2022

Autism spectrum disorder (ASD) is a highly heterogeneous disorder, yet transcriptomic

profiling of bulk brain tissue has identified substantial convergence among dysregu- Significance
lated genes and pathways in ASD. However, this approach lacks cell-specific resolution.
We performed comprehensive transcriptomic analyses on bulk tissue and laser-capture We present a comprehensive
microdissected (LCM) neurons from 59 postmortem human brains (27 ASD and 32 assessment of neuronal cell-type-
controls) in the superior temporal gyrus (STG) of individuals ranging from 2 to 73 years specific gene expression and
of age. In bulk tissue, synaptic signaling, heat shock protein-related pathways, and RNA alternative splicing changes in
splicing were significantly altered in ASD. There was age-dependent dysregulation of ASD cortex, directly comparing
genes involved in gamma aminobutyric acid (GABA) (GAD1 and GAD2) and glutamate RNA-seq results from bulk tissue
(SLC38A1) signaling pathways. In LCM neurons, AP-1-mediated neuroinflammation with isolated neurons. We
and insulin/IGF-1 signaling pathways were upregulated in ASD, while mitochondrial
observe strong signatures of cell
function, ribosome, and spliceosome components were downregulated. GABA synthesiz-
stress and neural-immune/
ing enzymes GAD1 and GAD2 were both downregulated in ASD neurons. Mechanistic
modeling suggested a direct link between inflammation and ASD in neurons, and pri- inflammatory pathway activation
oritized inflammation-associated genes for future study. Alterations in small nucleolar present within ASD neurons—a
RNAs (snoRNAs) associated with splicing events suggested interplay between snoRNA signal that is typically attributed
dysregulation and splicing disruption in neurons of individuals with ASD. Our findings to astrocyte/microglial
supported the fundamental hypothesis of altered neuronal communication in ASD, populations. Our findings also
demonstrated that inflammation was elevated at least in part in ASD neurons, and provide further evidence for the
may reveal windows of opportunity for biotherapeutics to target the trajectory of gene hypothesized imbalance of
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

expression and clinical manifestation of ASD throughout the human lifespan. excitatory to inhibitory neuronal
activity in the brains of
ASD | transcriptome | neuron-specific
individuals with ASD. Moreover,
Autism spectrum disorder (ASD) defines a heterogeneous set of complex neurodevelop- we find that the transcriptomic
mental disorders affecting 1 in 54 children in the USA according to current estimation architecture of ASD interacts
(1, 2) and confers lifelong challenges. ASD is characterized by difficulties with social substantially with age, thus
communication as well as a repetitive, restricted repertoire of behaviors and interests (3). revealing windows of opportunity
Population, family, and twin studies all indicate a strong genetic component contributing for treatments that target specific
to risk for ASDs (4, 5), with heritability estimates of ~70% (6). However, the genetic molecular pathology.
causes and pathophysiology of ASD are varied and often complex.
Despite this heterogeneity, transcriptomic analyses of postmortem human brain have
elucidated substantial convergent molecular-level pathology associated with idiopathic
and syndromic forms of ASD (7–13). Multiple studies have profiled the transcriptomes
of postmortem brain regions from individuals diagnosed with ASD (7, 8, 11, 13),
including the temporal cortex implicated due to its critical importance in speech and
language function (7, 8, 11, 13). The most consistent findings include disruption of
neuronal/synaptic activity and activation of innate immunity/glial markers (7, 8, 11). Author contributions: B.P.A., B.S., and C.M.S. designed
Alternative splicing and non-coding RNAs have also been shown to be dysregulated in research; P.Z., A.O., M.J.G., B.P.A., B.S., and C.M.S.
performed research; P.Z., A.O., M.J.G., and B.S. analyzed
ASD brains (8). data; M.J.G., B.S., and C.M.S. supervised the study; and
Most previous transcriptomic studies, however, profiled homogenate brain tissue and P.Z., B.P.A., M.J.G, B.S., and C.M.S. wrote the paper.

have therefore been unable to pinpoint the underlying specific cell types in which gene The authors declare no competing interest.

expression is altered. Single-nucleus RNA-sequencing (sn-RNAseq) datasets have started This article is a PNAS Direct Submission.

to be generate in postmortem ASD cortex (12, 13), identifying substantial changes in Copyright © 2023 the Author(s). Published by PNAS.
This open access article is distributed under Creative
upper-layer excitatory neurons and microglia, consistent with observations from bulk Commons Attribution License 4.0 (CC BY).
tissue. As such sn-RNAseq datasets currently primarily profile the 3′ end of highly 1
To whom correspondence may be addressed. Email:
expressed genes within each cell, these data characterize neither lowly expressed coding michael.gandal@pennmedicine.upenn.edu, bsstamova
@ucdavis.edu, or cschumann@ucdavis.edu.
and noncoding genes, nor splicing alterations that may contribute to altered neuronal 2
M.J.G., B.S., and C.M.S. contributed equally to this work.
function in ASD.
This article contains supporting information online at
Here, we performed the systematic study using transcriptomic profiling to directly https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.​
compare both bulk cortical tissue and laser capture microdissected (LCM) neurons from 2206758120/-/DCSupplemental.
anatomically well-defined superior temporal gyrus (STG) samples from 59 subjects (27 Published March 2, 2023.

PNAS 2023 Vol. 120 No. 10 e2206758120 https://doi.org/10.1073/pnas.2206758120 1 of 11

 with ASD and 32 controls) ranging from 2 to 73 years of age
(Fig. 1 and Dataset S12). The STG modulates language processing
and social perception, thereby playing a critical role in integrating
a breadth of information to provide meaning to the surrounding
world (14). Structural and functional imaging studies have long
implicated STG in ASD (14, 15); however, molecular-level
changes in neurons remain unknown. This study aimed to identify
neuron-specific transcriptomic changes in ASD brain by identi-
fying differentially expressed genes, differential splicing (DS)
events, age-related gene expression changes across the lifespan,
and co-expression networks to reveal gene modules altered in
ASD. We also applied mechanistic modeling approaches to

A
Ground tissue for
bulk analysis
3A
Total RNA extraction
Superior temporal gyrus 4 and QC
sampled from Tissue flash frozen
32 Controls in liquid N2

1 27 ASD
2 Embedded in OCT
for LCM
3B

Prepare cDNA libraries,

5 QC, and pool

Sectioned on cryostat Placed on PEN membrane Neurons (100/sample)

to 12 µm slides and stained captured directly into tubes
by laser capture microscope DNA sequencing
with HistoGene
(50M paired-end
3C 3D 3E 6 150 bp reads)
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

7 Data analysis pipeline

B Diagnosis Age
1.00
Sex
30 F
60
0.75
20
count

Age

40 0.50
10
20 0.25 M

0 0 0.00
Control Autism Control Autism Control Autism

PMI RIN
8

30
6
Hour

RIN

20
4
10

2
Control Autism Control Autism

Fig. 1. Overview of Experiment Design, Data Analysis Pipeline (A), and Study Cohort (B).

2 of 11 https://doi.org/10.1073/pnas.2206758120 pnas.org
 pinpoint pathways and genes directly linked to ASD in neurons. comprehensively characterized differential gene expression (DGE)
(Fig. 1). and local splicing alterations in ASD. After adjusting for known
covariates and correcting for multiple comparisons, we found 194
Results differentially expressed genes between individuals with ASD and
controls (FDR < 0.05). Of these, 143 were upregulated and 51
Global Gene Expression Changes in ASD STG. RNA sequencing were downregulated (Fig. 2A and Dataset S1), with a median
was performed on bulk tissue STG of 59 human brains, 27 absolute fold change of 1.45 (range 1.11 to 4.04, Fig. 2A). We
from individuals with ASD and 32 from neurotypical controls, observed significant concordance between our DGE results and
ranging from 2 to 73 years of age. Following quality control, we previous data of the same region from different samples (13)

A B
Number of DE genes ( FDR < 0.05)

Disease effect
25 Block-M32

*
*

*
-0.1 0 0.1
20 Block-M14 beta

*
*
*
Block-M3 GWAS enrichment

*
*

*
*
15 No. of genes upregulated: 143
Block-M19 0 0.5 1

*
*
*
-log10FDR
10 Block-M10

*
*
*
*

*
*
*
*
*
*
*
*
*
*
Cell type enrichment
5 Block-M31

*
*
*

*
*

*
0 1 2 3
Block-M1 -log10FDR

*
*

*
*
*
*
*

*
*
0

OPC

Microglia
InNeu

LGD+Missense
Presynaptic
Oligo

Endo
Astro

LGD DNMs
ExcNeu

Postsynaptic

pLI>0.99
Module-list overlap,

Syndromic ASD
Vulnerable
Constrained
ASD GWAS
Per
ASD expression

FMRP Target
CHD8 targets
5 permutation test

No. of genes downregulated: 51

10 0 5 10 15
-log10FDR
1.0 1.5 2.0 2.5 3.0 3.5 4.0
RNA-seq Fold Change

C
6 RGS8
MGA

SPEN
CRYAB

HSPB1
D chaperone-mediated protein folding
DNA packaging complex q value
FXR1 PLOD3
GAN
BAG3
nucleosome 0.018
NEMF PIGT SLC16A1SNX31HSPA1A response to unfolded protein 0.015
CLUL1
PCBP1-AS1 TRA2A HIBCH
BCOR
NKAIN1
LINC01535 CPEB1-AS1 extrinsic apoptotic signaling pathway
C20orf27 ZBTB22 CRKL
DNAJB4 SPR
regulation of intracellular pH 0.012
NR2F2-AS1 CDKN2D XPNPEP3 SAV1 HIST2H2BD HSPA1B CP
NR2F2
CDC7 KIAA1614
RBM48 JAM3 ST6GALNAC4P1 unfolded protein binding 0.009
4 MYO1E
myelination
-log10 p value

KCNH3 PTGES3 DNAJB1

regulation of transcription in response to stress
cellular response to unfolded protein
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

HSPB8
KCNIP1
synaptic transmission, glutamatergic
neurotransmitter receptor complex overlap
Golgi lumen 20
2 collagen trimer 30
multicellular organismal response to stress 40
neurotransmitter receptor activity 50
extracellular matrix structural constituent 60
G protein-coupled peptide receptor activity 70
excitatory postsynaptic potential
0 ammonium ion binding
-2 -1 0 1 2 -2 -1 0 1 2
log2 FoldChange NES

E 14
F EPB41L3
12
10 Significant DS: 308

13
GAD1 expression

GAD2 expression

SEPT8 APBA2
in block tissue

in block tissue

MED4 ANKRD20A3 COG1

8
observed

CD47 MARCH7 ROBO3

9 12 Diagnosis CLASP2 CNTNAP1
Control NALCN IQSEC1 CLASP2
PIK3AP1 EWSR1
Autism
MACF1
11
8 4

10
Diagnosis * Age interaction FDR = 0.026
7 Diagnosis * Age interaction FDR = 0.047
9 0
0 20 40 60 0 20 40 60 0 1 2 3 4 5
Age Age expected

Fig. 2. Transcriptomic Difference Between ASD Cases and Controls in Bulk Tissue STG. (A) Distribution of fold-change of differential expression for 194
differentially expressed genes. Case/control fold-changes for upregulated genes are plotted in gold (N = 143, positive values) and control/case fold-changes for
downregulated genes in blue (N = 51, negative values). (B) Co-expressed gene modules that were significantly disrupted in ASD. Modules were hierarchically
clustered by module eigengene. Module-diagnosis associations were shown on the right of each module (*FDR < 0.05). Additional enrichment analyses were also
shown for each module, including: Enrichment for ASD GWAS common variants (33)(*FDR < 0.1); Enrichment for major CNS cell types (16)(*FDR < 0.05); Enrichment
against literature-curated gene lists (*FDR < 0.05, see SI Appendix, SI Methods for details of the gene lists). Per, pericytes; OPC, oligodendrocyte progenitor cells;
InNeu, inhibitory neuron; ExcNeu, excitatory neuron; Oligo, oligodendrocytes; Endo, endothelial cells; Astro, astrocytes. (C) Volcano plot showing significantly
up- (gold) and down-regulated (blue) genes (FDR < 0.05). Genes discussed in the main text are colored red. (D) Functional enrichment of differentially expressed
genes in ASD cases compared to controls. Top 10 significantly enriched up- and down-regulated categories were shown. The color of each dot reflects FDR-
corrected q-value, and the size of each dot reflects the number of overlapped genes between our gene list and the corresponding GO category. NES, normalized
enrichment score. (E) Age trajectory of GAD1 (Left) and GAD2 (Right) gene expression, stratified by ASD diagnosis. The expression levels were on the log2CPM
(counts per million) scale. (F) Quantile-quantile plot of observed P-values vs. expected P-values for differentially spliced intron clusters. Significant DS events
(FDR < 0.05) were colored red, and overlapping gene names were labeled for top clusters.

PNAS 2023 Vol. 120 No. 10 e2206758120 https://doi.org/10.1073/pnas.2206758120 3 of 11

 (SI Appendix, Fig. S1, Spearman ρ = 0.37 for t statistics among
all genes, P value < 10−16).
Functional and pathway enrichment analyses indicated an
over-representation of heat-shock proteins (HSPs) and HSP-
related chaperones, which were upregulated in ASD subjects. This
included HSP70 family members HSPA1A and HSPA1B; HSP40
family members DNAJB1 and DNAJB4; small HSP20 family
members HSPB1 and HSPB8; and HSP-binding chaperons BAG3
and PTGES3 (Fig. 2 C and D). HSPs are involved in stress
response, immune activation, and inflammation(17, 18), all of
which were upregulated in ASD postmortem brain (7).
Downregulated genes were mainly enriched in pathways related
to synaptic function (Fig. 2D), consistent with previous findings
(7). Notably, two important voltage-gated potassium channel-
related genes KCNH3 and KCNIP1 were among the most down-
regulated (Fig. 2C), which may relate to disrupted neuronal excit-
ability hypothesized in ASD (19, 20).
As age-dependent expression alterations have been reported in
ASD brain (21), we employed an analytical model accounting for
age and the interaction between age and diagnosis. Fourteen genes
showed age-dependent DGE between ASD and control (Dataset
S2). Gene set enrichment analysis (GSEA) indicated that genes
with significant diagnosis-by-age interaction were enriched in
immune/inflammation pathways and synaptic-related pathways.
(SI Appendix, Fig. S9).
Interestingly, genes involved in gamma aminobutyric acid (GABA)
synthesis (GAD1 and GAD2) (22) were downregulated in ASD only
during late adulthood (Fig. 2E). This may indicate an age-dependent
dysregulation of GABA signaling in ASD neurons, or a decrease in
the proportion of GABAergic neurons in ASD brains (23).
Given mounting evidence suggesting potential mechanistic
overlap between neurodevelopmental and neurodegenerative dis-
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.
orders, particularly with respect to genes involved in synapse and
brain connectivity (24), we evaluated the overlap between our
ASD findings with published results in Alzheimer’s disease (AD).
Using results from Mount Sinai/JJ Peters VA Medical Center Brain
Bank (MSBB) cohort (25), we demonstrated that the changes we
detected in ASD were significantly overlapped with transcriptomic
alterations observed in AD from the same brain region (Odds
Ratio = 1.6, P = 1.9 × 10−8, SI Appendix, Fig. S8).
DS events in the bulk tissue transcriptome were evaluated using
LeafCutter (26). Among 35,505 intron clusters identified by
LeafCutter, 308 clusters (297 unique genes) showed significant
DS between ASD patients and controls (FDR < 0.05). The 297
genes did not show significant functional enrichment (Fig. 2F and
Dataset S5).
To place subtle changes across the ASD STG transcriptome
into a systems-level context, we performed weighted gene corre-
lation network analysis (WGCNA) to build gene co-expression
networks (27), identifying 31 modules of co-expressed genes
(Methods and Dataset S3). Seven modules significantly associated
with ASD diagnosis, two of which were strongly enriched for
ASD-associated genetic risk factors (Modules Block-M1 and
Block-M10, Fig. 2B).
Module Block-M1 was upregulated in ASD STG and its gene
members were enriched in RNA splicing and mRNA metabolic
pathways (Dataset S4). Notably, significantly upregulated HSPs
were also members of the Block-M1 module (Dataset S3). HSPs
contribute to RNA splicing during stress (28). Downregulated
modules in ASD were mostly enriched for synaptic functions
(Block-M3, Block-M10, Block-M14, Block-M19, Block-M31;
Dataset S4). Cell-type enrichment analysis also indicated these
downregulated modules were enriched in marker genes for both
4 of 11 https://doi.org/10.1073/pnas.2206758120
excitatory and inhibitory neurons (Fig. 2B), suggesting a broad
disruption of neuronal and synaptic processes in ASD STG.
Genes in the upregulated module Block-M1 and one downreg-
ulated module (Block-M10) were enriched in high-confidence
ASD risk loci (29, 30), mutationally constrained (31), and highly
intolerant to mutations (pLI > 0.99) genes (32), as well as regu-
latory target genes of CHD8, which has clear links to at least a
subset of ASD cases (33) (Fig. 2B, see SI Appendix, SI Methods for
the details of all curated, hypothesis-driven gene sets. Dataset
S14). Many hub genes for module Block-M10 encoded synaptic
proteins (SI Appendix, Fig. S2 and Dataset S3). This module was
also enriched for ASD common risk alleles from ASD GWAS data
(34). Together this suggested a causal role of synaptic dysfunction
in ASD etiology.
Neuron-Specific Gene Expression and Splicing Alterations in ASD
STG. To provide cell-type specificity for the observed transcriptomic
changes, we performed laser capture microdissection to capture
neurons using STG sections taken from the same subjects profiled
using bulk RNA-seq (SI Appendix, Figs. S6 and S7). We then
interrogated ASD-associated gene expression and splicing alterations
using the same bioinformatic pipelines as above. Across 13,458
neuron-expressed genes, 83 were significantly differentially expressed
between ASD subjects and controls at FDR <0.05, of which 52 were
upregulated and 31 downregulated (Fig. 3A and Dataset S6). Median
absolute fold change in expression between subjects with ASD and
controls was 2.48 (range 1.29 to 9.72; Fig. 3A). The concordance of
neuronal DGE with bulk tissue DGE was low (Spearman ρ = 0.18
for t statistics across all genes, SI Appendix, Fig. S3), suggesting our
analysis captured ASD signatures unique to neurons.
Upregulated genes in ASD neurons were highly enriched in
pathways related to growth and differentiation (Fig. 3E).
Specifically, the AP-1 transcription factor complex components
(35) FOS, JUN and JUNB were all upregulated in ASD neurons,
among other growth/differentiation regulators such as SOX9,
S1PR1, and PPP1R16B (Fig. 3D). The AP-1 transcription factor
complex is known to regulate many downstream biological pro-
cesses (35). Indeed, we found upregulated B cell signaling adap-
tor gene BCL10 and NFκB inhibitor delta gene NFKBID in
ASD neurons (Fig. 3D). Up-regulation of both NFKBID and
AP-1 point to dysregulated inflammation in ASD neurons. In
addition, the inward-rectifier potassium ion channel gene
KCNJ2 was also upregulated in ASD neurons (Fig. 3D).
Interestingly, both KCNJ2 and AP-1 subunit FOS were involved
in regulating excitability and plasticity at the cholinergic synapse
(36–38).
Downregulated genes in ASD neurons are primarily enriched in
mitochondrial function and oxidoreductase activity (Fig. 3E).
Specifically, comparing to bulk tissue STG, more subunits of the
NADH:ubiquinone oxidoreductase (complex I) were downregulated
in neurons, and their effect sizes were larger (SI Appendix, Fig. S4).
Our results provide evidence that compared to bulk tissue, mito-
chondrial dysfunction is much more profound in STG neurons.
While LCM captured both excitatory and inhibitory neurons,
we note that GAD1 and GAD2 genes are among the most down-
regulated in ASD neurons (Fig. 3D). The coordinated down-reg-
ulation of both GABA synthesizing enzymes suggest that the level
of GABA neurotransmitter may be decreased in ASD neurons,
providing support to the excitation to inhibition (E/I) imbalance
hypothesis of ASD (19, 20, 39).
By testing the interaction between diagnosis and age, three
genes (HTRA2, aka OMI; ZNF765; and PCDHB18P) showed
age-dependent differential expression in ASD neurons (Dataset S7;
pnas.org
 A B C
7.5
Number of DE genes ( FDR < 0.05)
10 FRYL
L
Diagnosis * Age interaction FDR = 0.009 12 Significant DS: 1292
MED23
No. of genes upregulated: 52 ANK2 CCNDBP1
CAMK1D PLEKHG3 QRSL1

HTRA2 expression
5 5.0 CFAP69 BAG4 CNOT1
ZNF384 USP53
8 NUDT6 CEP164

observed
in neuron
Diagnosis VWA3A OCEL1L1
1
RBBP8
0 2.5 Control
Autism
neuron
4 block tissue

5 0.0
No. of genes downregulated: 31

-2.5 0
1 2 3 4 5 6 7 8 9 10 20 40 60 0 1 2 3 4 5
RNA-seq Fold Change Age expected

D 8
E
growth factor binding
ELL
endothelium development
LAMB2
kidney morphogenesis
KCNC2 DAB2
CSRNP1
endothelial cell differentiation
AL365209.1 cytokine binding q value
6 TOB2
RNU1-67P UMAD1 INPP5A TACO1
AC004890.2
ureteric bud development 0.030
GAD2 FAM216A SPINDOC
KANK2
nuclear receptor transcription coactivator activity 0.025
RGS5 RPL32 SLC45A4
branching morphogenesis of an epithelial tube
-log10 p value

JAM2 BCL10
GAD1 PDHX TMCC2 JUNTPRA1 ESRRA 0.020
RFC3
NDUFA4 ROBO2
PEAK1
PPP1R16B RASD1 JUNB MT-TT response to glucocorticoid
4 NPIPB11 RPS21 FDFT1 ATP5F1B
SLC6A8
KCNJ2 AC069120.1
FAM107A TLE3 C3orf33 regulation of cytokine-mediated signaling pathway 0.015

SOX9
FOS structural constituent of ribosome
S1PR1 NFKBID
protein targeting to ER
oxidoreductase activity, acting on NAD(P)H, quinone overlap
mitochondrial respiratory chain complex I assembly 20
2 Sm-like protein family complex 40
spliceosomal snRNP complex 60
respiratory chain
inner mitochondrial membrane protein complex
respiratory chain complex
0 oxidoreductase complex
-2 0 2 -2 -1 0 1 2
log2 FoldChange NES

Fig. 3. DGE and DS Between ASD Cases and Controls in LCM Neurons from STG. (A) Distribution of fold-change of differential expression for 83 differentially
expressed genes. Case/control fold-changes for upregulated genes are plotted in gold (N = 52, positive values) and control/case fold-changes for downregulated
genes in blue (N = 31, negative values). (B) Age trajectory of HTRA2 gene expression, stratified by ASD diagnosis. The expression levels are on the log2CPM (counts
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

per million) scale. (C) Quantile–quantile plot of observed P-values vs. expected P-values for differentially spliced intron clusters. Significant DS events (FDR < 0.05)
were colored red, and overlapping gene names were labeled for top clusters. DS results from bulk tissue were also plotted for comparison. (D) Volcano plot showing
up- (gold) and down-regulated (blue) genes. Genes discussed in the main text are colored red. (E) Functional enrichment of differentially expressed genes in ASD
cases compared to controls. Top 10 significantly enriched up- and downregulated categories are shown. The color of each dot reflects FDR-corrected q-value,
and the size of each dot reflects the number of overlapped genes between our gene list and the corresponding GO category. NES, normalized enrichment score.

no pathway enrichment was detected for neuronal genes with significantly upregulated in ASD neurons, while one module was
significant diagnosis-by-age interaction). For example, age trajec- downregulated (Fig. 4A).
tories of serine peptidase HTRA2 were opposite in ASD brains The upregulated neu-M5 co-expression module was highly
compared to controls (Fig. 3B). In healthy brains, the expression represented by the DGE analysis signal. Upregulated genes
of HTRA2 was much higher before age 30 and decreases with age, JUN, JUNB, and NFKBID were all hub genes of neu-M5 mod-
while its expression levels begin lower and increase with age in ule. Neu-M5 module also captured additional AP-1 subunits
ASD STG neurons. and interactors, such as FOSL2 (40) and IRF3 (41, 42). Neu-
To further evaluate potential overlap between ASD and neuro- M5 module was enriched in immune response pathways, pro-
degenerative processes in neurons, we compared our neuronal viding further evidence that AP-1-mediated neuroinflammation
findings with results from MSBB. Changes in ASD neuronal was elevated in ASD neurons (Fig. 4C and Dataset S10). Hubs
transcriptome were also significantly overlapped with transcrip- of Neu-M5 also contained multiple ion channel-related genes,
tomic alterations observed in AD from the same brain region (odds such as sodium ion channel gene SCN1B, potassium channel
ratio = 1.4, P = 0.01, SI Appendix, Fig. S8). genes KCNJ2 and KCNJ10, and solute carrier gene SLC40A1
We also quantified local splicing events in the neuronal tran- (Fig. 4C and Dataset S9). Coordinated upregulation of various
scriptome. After adjusting for multiple testing, LeafCutter iden- ion channels suggested that membrane transport was activated
tified 1,292 significant differential spliced intron clusters (1,177 in ASD neurons, consistent with heightened excitability. Neu-
unique genes) out of 17,250 total intron clusters at FDR < 0.05 M5 was significantly overlapped with module M16 from
(Fig. 3C and Dataset S8). No functional enrichment was Voineagu et al. (7). M16 was also enriched in immune/inflam-
observed for the 1,177 genes. We observed more disruptions in matory response and was up-regulated in ASD (7). Our data
local splicing events in ASD neurons than in bulk tissue (308 refined our understanding of the neuroinflammatory changes
DS out of 35,505 events in bulk tissue, 1,292 DS out of 17,250 in ASD to include a neuronal component. Additionally, down-
events in neurons; P < 2 × 10−16 test of proportions). regulated neu-M17 module was enriched in mitochondrial
function and contained the most differentially expressed mito-
Neuron-Specific Networks Pinpoint Subtle Changes in the chondrial genes, such as ATP synthase subunits ATP5F1B and
Neuronal Transcriptome in ASD. Co-expression network analysis ATP5PF (Fig. 4F and Dataset S9).
on neuronal data identified 18 modules, each containing between Neuronal co-expression networks further captured signals that
101 and 998 co-expressed genes (Dataset S9). Four modules were were not detected by DGE analysis. Neu-M6 module was

PNAS 2023 Vol. 120 No. 10 e2206758120 https://doi.org/10.1073/pnas.2206758120 5 of 11

 A B Excitatory neurons Inhibitory neurons
*

Neu-M13
Neu-M6
Neu-M7
Neu-M4
Neu-M5
Neu-M3

Neu-M9

Neu-M2
Neu-M8
Neu-M10

Neu-M12

Neu-M14
Neu-M15
Neu-M11
Neu-M16

Neu-M17
Neu-M18
Neu-M1
Disease effect 6 * Neuron Modules
* * ** *

Z-score
ASD expression -0.1 0 0.1
Neu-M5
beta
ASD_GWAS
* 4 * Neu-M11
Postsynaptic
* * ** * GWAS enrichment
* Neu-M17
Presynaptic
* ** 0 Neu-M16
* * **
0.5 1
LGD+Missense
-log10FDR 2
LGD DNMs
* * * * Neu-M6
pLI>0.99
* * ** * ** Module-list overlap,
Vulnerable
* * * * permutation test 0
Constrained
* * ** *
FMRP Target
* * * ** *

Ex5b
Ex6b

In1b

In4b
In6b
Ex3e

Ex6a

In1a
In1c
In4a
In6a
0 5 10 15
* * * **

Ex1
Ex2
Ex4

Ex8
Ex9

In3

In7
In8
CHD8_targets -log10FDR
Syndromic ASD

C Neu-M5
D Neu-M6
E Neu-M16
signal release from synapse
sequence-specific DNA binding regulation of metal ion transport
neurotransmitter secretion
positive regulation of myeloid leukocyte regulation of cell-matrix adhesion
cytokine production in immune response calcium ion regulated exocytosis
positive regulation of intracellular signal transduction regulation of carbohydrate biosynthetic
microtubule
positive regulation of immune effector process regulation of calcium ion transport
vesicle-mediated transport in synapse
nucleobase-containing small molecule interconversion positive regulation of smooth muscle cell migration
synaptic vesicle cycle
negative regulation of pri-miRNA transcription
transmembrane transporter activity regulation of axon extension involved in axon guidance
channel activity hexose transmembrane transport
0 1 2
0.0 0.5 1.0 1.5 0 1 2 -log10FDR
-log10FDR -log10FDR
AC104024.2 ERGIC1 ARHGAP39
DOCK1 AHDC1 TNK2
KCNH8 GLRX CAMK1D
RASSF8-AS1 RAI1 IQSEC3
POLD2 GPRC5B SLC6A8
DIP2A
CTPS2 SENP3 ENDOD1 AMMECR1LP1
KCNJ10 SORBS1 LAS1L WIPF3 ZNF83 MLLT6
LIX1 SCAF1
POU2F2 HMGA1 UNC5A MEGF8 PSD
NFKBID TACO1 IRF3 AL391684.1 PTK2B
CSRP1 SCN1B DHX30 PIP5K1C
EPB41L1 SIPA1L3 LRRC4B
PCDHGB4 NME6 SND1 NSD1 EPN1
CPLX2 TOM1L2
SLC7A11 ABLIM2
UBE2L6 IGF1R NAPRT LLGL1
F8 LINC-PINT HCFC1
FGF1 TNR SAMD4B PPP1R16B POM121
LRP4 SLC40A1 S1PR1 PEAK1 CIT TTC28 GNA11
BHLHE40 HECTD3 CBX6
SLC39A14 KSR2 ANXA11 KRBA1
SOX2 MT-TT AJAP1 PCDHGB
NHSL2 KCNAB2 INPP5E
USP3 FGFR2 CAMK2A
ATP1A2 IGFBP5 PTOV1 PRRT2 CLIP2
LINC00476 ELL BCL10 PRDM11 TAMM41 SYNGR1
ELK1
ZBTB7C INPP5A ARID1A WFS1 MAP1S PCSK1N
JUN KCNJ2 AC097376.2 NCLN
ALDH6A1 UBE2H SNPH USP20
TLE3 FBXL20 FAM107ASPECC1 RNF130 SIRT7
AC138393.3 PROX1 AC016831.4 CAMTA2
TOB2 FOSL2 REPIN1 IGLON5
AC004890.2BAHD1AL080317.3 RANGAP1 SCAMP5
ADGRV1 PLOD1 DDN CYHR1
SGMS2 IRS2
IBA57 BTG2 CADM3 PACSIN1 PNKD
POU3F3
LINC02210
MCU
KCTD15

F mitochondrial membrane
G Neu-M18 Neu-M7 Neu-M9 H BCL10
AC004890.2

Neu-M17 Neu-M14
MT-TT
TLE3
proteasome core complex
ZBTB7C
oxidative phosphorylation TACO1
Neu-M12 Neu-M17 Neu-M2 Neu-M3 IBA57
mitochondrial translation USP3

mitochondrial inner membrane KCNJ2

GALNT2 S1PR1
farnesyl diphosphate metabolic process Neu-M13 Neu-M6 Neu-M8 Neu-M4 BAHD1
TOB2
antigen processing and presentation of ELL
endogenous peptide antigen via MHC class I JUN
SDC2
0.0 0.5 1.0 1.5
TLNRD1
-log10FDR Neu-M1
Neu-M5 Neu-M11 Neu-M15 Neu-M16 Neu-M10 INPP5A JUNB
SFT2D1
MARK1 LINC00476 GTF2IRD2B
COX7C
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

ECHS1
BUB3
RPLP2
ASD
PDZD11 ZWILCH

I
ANAPC7
MLLT11 CCT7
HSBP1 HELLPAR
RBM11 FDPS
Upstream regulator

PAPOLG
RFC3 DDX20
PSMA1 dopamine
KCNAB1
PSMB3 IFIT1
(predicted)

CDH7 TMEM70
NTN4
MRPS9 KLHL5

NAALAD2 EPHA7
TFG ATP5F1B
Activated
C6orf120 COA7
ATG5 CBX1
beta.estradiol DRD2 CREB1 Inhibited
GATC Pka ELK1
MRPL3
ATP5PF
TMEM14A C1orf52 Affected
TXNDC16 NDUFB6 DCUN1D5 TRHDE
CYB5R4
AC104083.1 CISD2
FOXO1 FOXO3 STAT5B CREM RNA.polymerase.II
LAMTOR3 RELA MEF2D Creb ATF1 HIF1A ESR1
RNF170
C2orf69 Direct interaction
Indirect interaction
Differentially expressed gene

NDUFA4 RASD1 PDGFB

GAD1 JUN JUNB FOS FOSL2
SLC7A11
(observed)

BCL10 SOX9 CSRNP1 ALAS1 NF1 ERBB4

SLC1A3 GAD2 NFKBID
Differential expression Log2FC

ESRRA EGR4 NPAS4 GADD45B ATP5F1B

Transporter GABA synthesis -3 0 3

Transcription regulator Mitochondrial

Fig. 4. Co-expression and Mechanistic Network Analysis of the ASD Neuronal Transcriptome. (A) Hierarchical clustering of neuronal gene co-expression
modules by module eigengenes. Module–diagnosis associations were shown below each module. Enrichment for ASD GWAS common variants is shown for
each module. Enrichment against literature-curated gene lists is shown on the bottom. (B) Module enrichment for neuron subtypes. Expression profiles of
neuron subtypes were obtained from ref. 16. Red asterisks indicate significant enrichment. (C–F) Functional enrichment (Top) and top 50 hub genes (Bottom) for
module Neu-M5 (C), Neu-M6 (D), Neu-M16 (E) and Neu-M17 (F). Edges represent co-expression (Pearson correlation > 0.5). Co-expressed partners with evidence
of protein-protein interaction (PPI) were connected by solid black lines. PPI data was compiled from well-characterized PPI databases (SI Appendix, SI Methods).
(G) Bayesian network representing the relationships among modules (ellipse) and ASD diagnosis (diamond). The thickness of the arrow is proportional to the
number of times that a connection was detected during a bootstrap of 500 times. (H) The estimated gene regulatory network (Bayesian network) for 21 hub
genes in Neu-M5. (I) Dopamine network built by IPA from neuron DEGs (FDR < 0.1, 225 genes). A network of 17 upstream regulators (orange diamonds) were
predicted to drive the changes of 74 observed target genes (23 of which were shown based on node connectivity ≥ 2, ellipse). ELK1 was differentially expressed
in neurons, and it was also predicted as an upstream regulator.

upregulated in ASD, and among its hub genes were several insulin Neu-M16 was enriched in synaptic functions (Fig. 4E and Dataset
signaling pathway components, including insulin-like growth factor S10). Further, cell-type analysis showed that Neu-M16 was also
(IGF) receptor IGF1R, IGF binding protein IGFBP5, insulin recep- highly enriched in excitatory neurons (Fig. 4B), with CAMK2A
tor substrate IRS2 as well as CBL-associated SORBS1 (Fig. 4D and and CAMK2B among its hub genes (Dataset S9). The upregulation
Dataset S9). Our results provided direct molecular-level evidence of Neu-M16 suggested elevated excitatory signal in ASD
that insulin signaling was altered in ASD neurons. neurons.
Among all five significantly disrupted modules, none were We further tested whether significantly disrupted neuronal
enriched for ASD common variants and only one upregulated modules were enriched in any neuron subtypes. Upregulated neu-
module (Neu-M16) showed enrichment in highly confident ASD ronal modules in ASD were only enriched in excitatory neuron
risk genes, as well as in several other curated gene sets (Fig. 4A). subtypes, while enrichment of inhibitory neurons was only

6 of 11 https://doi.org/10.1073/pnas.2206758120 pnas.org
 observed for downregulated modules (Fig. 4B). This provides
additional evidence for altered neuronal activity in ASD neurons,
consistent with the findings in our DGE analysis.
To gain mechanistic insights from our neuronal data, we used
Bayesian network inference (43–45) to distinguish between
direct module-trait associations and indirect module-trait cor-
relations. Our Bayesian network contained 19 nodes, including
18 neuronal co-expressed gene modules represented by module
eigengenes, plus a binary node representing ASD diagnosis. Our
results indicated that Neu-M5 was directly associated with ASD,
conditioned on all other modules (Fig. 4G). This provided fur-
ther evidence that neuroinflammation was directly associated
with ASD in neurons.
We further used Bayesian network to prioritize among hub
genes of Neu-M5 module for future validation. We focused on
21 genes within Neu-M5 that were also significant differentially
expressed genes (DEGs). The Bayesian network learned from these
21 hub genes is shown in Fig. 4H. Genes with the highest degree
of connectivity in this network included BCL10, ELL, and
GTF2IRD2B, which can be potential targets for functional
evaluations.
As an orthogonal approach, we identified the upstream regulators
and built mechanistic networks in Ingenuity Pathway Analysis
(IPA® ) with the differentially expressed genes in captured neurons
(FDR < 0.1, 225 genes, Dataset S13). Dopamine was predicted as
a top upstream regulator with the mechanistic network presented
in Fig. 4I. This dopamine network contained 17 upstream regulators
predicted to drive the observed changes of 74 target genes, including
AP-1 subunits JUN, JUNB, FOS, and GABA synthetase GAD1
and GAD2. Moreover, there were seven genes (BCL10, JUN, JUNB,
KCNJ2, ELL, S1PR1, and SDC2) from this mechanistic network
that overlapped with the 21 genes identified by the Bayesian net-
work analysis. This further highlighted the involvement of these
genes in ASD, since they were identified independently by different
modeling approaches.
Small Noncoding RNAs Are Selectively Downregulated in ASD Neurons
and Correlate with Altered Local Splicing. When investigating
the genes downregulated in ASD neurons more closely, we
noticed a striking pattern that 51 out of the 59 neuron-expressed
small nucleolar RNA (snoRNA) (46) genes were downregulated
in ASD neurons, and 13 were significantly downregulated at
P-value < 0.05 (Fig. 5A and Dataset S6). Dysregulation of snoRNAs
was not observed in bulk tissue (Dataset S1), and snoRNAs were
undetectable in a recent ASD single-cell study (12). snoRNAs are
involved in the modification and maturation of ribosomal RNAs
(rRNAs) and small nuclear RNAs (snRNAs) (47). Interestingly,
both ribosome and spliceosome components were among the most
downregulated in ASD neurons (Fig. 3E). Moreover, snRNAs were
also downregulated in ASD neurons, with 23 out of 24 snRNA genes

A 8 Neuon
B TFDP2
ANKRD28
DPP10
PCDH17
GRIA1
NDUFAF6
NPAS2
6 FMNL2
Pearson
PTPRN2
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.

LY6H Correlation
GRK4 1.0
-log10 p value

GCC2
ENTPD4 0.5
RAB3A
IPO13
4 SNORA23 MPHOSPH9
0.0
FDR = 0.05
SCYL2 -0.5
SNORA53 PDE4B
SNORA46 SCARNA5 TLN2 -1.0
FBXO9
SNORA26 GOT1
SNORD13 SNORA63 RAPGEF4
2 SNORA63 SNORA76
ARHGEF11
p = 0.05 TXN
SNORA40 SNORA13 SCARNA13
PPP4R3B
SNORD89 GRIA2
NEMF
ARIH1
SNORA74A
SNORA20
SNORA26
SNORD115-1
SCARNA3
SNORA71B
SNORA63
SNORD89
SCARNA5
SNORA53
SCARNA1
SNORA22
SNORA46
SNORA72
SNORA54
SNORD3A
SNORA35
SCARNA6
SCARNA21
SNORA12
SNORA23
SNORD15B
SNORA76
SNORA49
SNORA71D
SNORA13
SNORD13
SNORA42
SNORA80
SNORD3B-2

0
-2 0 2
log2FoldChange

C D
10.0 p = 0.0027
3
chr10:101163631-101165513

snoRNAs
7.5
snoRNA expression

SCARNA1
2
normalized PSI

SCARNA5
GOT1 Intron

SNORA12
5.0 SNORA13 1
SNORA23
SNORA46
2.5 SNORA53 0
SNORA63
SNORD15B
0.0 -1
SNORD89

-2.5 -2
-2 -1 0 1 2 3 Control Autism
GOT1 Intron chr10:101163631-101165513 PSI

Fig. 5. Coordinated Dysregulation of snoRNAs in ASD Neurons. (A) Volcano plot showing differentially expressed genes in ASD neurons compared to control.
snoRNA genes were colored red. (B) Significant correlation between snoRNA expression (x-axis) and intron PSI (y-axis). Introns are labeled with the name of
overlapping gene locus. Gene loci that are correlated with more than 3 snoRNAs were shown. (C) Scatter plot showing the correlation between GOT1 intron 5
PSI and expression levels of multiple snoRNAs across all neuron samples. Also shown were fitted regression lines with 95% CIs. Intron coordinates were based
on GRCh37. (D), PSI of GOT1 intron 5 is downregulated in ASD neurons.

PNAS 2023 Vol. 120 No. 10 e2206758120 https://doi.org/10.1073/pnas.2206758120 7 of 11

 Table 1. Summary of genes discussed in this manuscript
Noted Categories Genes
E/I signal CAMK2A*; CAMK2B*; GAD1*; GAD2*;
GOT1*
Growth / differentiation FOS*; FOSL2*; IRF3*; JUN*; JUNB*;
PPP1R16B*; S1PR1*; SOX9*
Heat shock BAG3†; DNAJB1†; DNAJB4†; HSPA1A†;
HSPA1B†; HSPB1†; HSPB8†;
PTGES3†
Immune / inflammation BCL10*; NFKBID*
Insulin signaling IGF1R*; IGFBP5*; IRS2*; SORBS1*
Ion channel KCNH3†; KCNIP1†; KCNJ10*; KCNJ2*;
SCN1B*; SLC40A1*
Mitochondrial ATP5F1B*; ATP5PF*
Age-associated GAD1†; GAD2†; HTRA2*; PCDH-
B18P*; ZNF765*
*
observed in STG neurons.
†
observed in bulk STG.
The category of each gene is based on what we discussed in this manuscript.
downregulated in ASD and 13 significantly downregulated at P value
< 0.05 (SI Appendix, Fig. S5 and Dataset S6) Table 1.
As snoRNAs and snRNAs are known to be critical regulators of
alternative splicing (48–51), and splicing alterations are strongly
implicated in ASD and observed in LCM-captured neurons, we next
examined alterations in splicing events that may be associated with
snoRNA dysregulation. We calculated the correlation between
snoRNA expression level and each local splicing event in neuronal
data, followed by FDR correction for multiple comparisons. We
identified 835 gene loci in neurons with at least one intron whose
percent spliced in (PSI) was significantly correlated with snoRNA
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.
gene expression (Dataset S11). Of these 835 intron clusters, 196
were significantly dysregulated in ASD neurons (Dataset S11).
Several intron clusters correlated with multiple snoRNAs and cor-
responded to genes involved in synaptic functions (Fig. 5B). For
example, GOT1 encodes the glutamic-oxaloacetic transaminase
known to function as an essential regulator of glutamate level (52).
PSI of intron 5 of GOT1 was highly correlated with the expression
level of multiple snoRNA genes (Fig. 5C). GOT1 intron 5 was also
differentially spliced between ASD and control (Fig. 5D). DS of
GOT1 gene may change the level of glutamate and thus leads to an
imbalance of E/I in neuronal communication in ASD neurons.
Discussion
Bulk Tissue Transcriptome Findings Reveal Downregulated
Neuronal and Synaptic Function Processes, Upregulation of
HSPs, and Unfolded Protein Response (UPR) in ASD Temporal
Cortex. Our bulk tissue analyses revealed a potential causal role
of downregulated neuronal processes and synaptic functions in
ASD etiology, consistent with findings from previous bulk-tissue
transcriptomic studies on the same brain region (7). Previous studies
also reported dysregulated alternative splicing events in ASD brain
(8, 53). DS analysis in bulk STG found several synaptic genes,
including CANCA2D1, CAMK4, CLASP2, CNTNAP1, EPHB1,
KALRN, NRXN3, SOS2 and SYNGAP1, differentially spliced in
ASD. SYNGAP1 isoforms have been shown to differentially regulate
synaptic plasticity and dendritic development (54). This further
signifies the importance of studying alternatively spliced isoforms
in ASD brain.
We also observed a coordinated upregulation of multiple HSPs
and HSP-related chaperones in ASD STG. HSPs can serve as acti-
vators and regulators of the immune system (18), and upregulated
8 of 11 https://doi.org/10.1073/pnas.2206758120
HSPs may induce immune responses in ASD brain. They also play
a role in facilitating alternative RNA splicing (28). Previous studies
found that both immune response and RNA splicing are upregulated
in ASD brain (7, 8, 11), and our results signify that upregulated
HSP-related pathways potentially contribute to these observations.
HSPs and HSP-related chaperones are normally induced in response
to stress. The upregulation of HSPs in ASD neurons may relate to
elevated endoplasmic reticulum (ER) stress since both UPR and
apoptosis are also upregulated in our ASD bulk data (Fig. 2D). ASD-
linked rare or de novo mutations in synaptic genes can lead to mis-
folded proteins and cause ER stress (55), itself coupled to heightened
inflammation and neurotoxic cell death (56). ER stress-related genes
are also dysregulated in the middle frontal cortex of subjects with
ASD (57). Our data suggest that ER stress is a major response to
ASD genetic mutations, and ER stress activates UPR, including the
production of HSPs and chaperones. UPR further induces multiple
downstream processes such as inflammation and immune response
(58). Limiting the effect of ER stress and UPR may be a promising
therapeutic avenue for ASD.
ASD Neuronal Transcriptome Reveals Upregulated
Neuroinflammation and Altered Neuronal Activity. We observed a
strong upregulation of AP-1 transcription factor components in ASD
neurons. AP-1 subunits FOS, JUN and JUNB were upregulated at
FDR < 0.05, and FOSL2 was upregulated at nominal P < 0.05. AP-1
regulated gene expression in response to various stimuli, including
cytokines, growth factors, stress signals, infections and inflammation/
neuroinflammation (35). In ASD neurons, AP-1 activation likely
induces broad inflammatory response, since several immune and
inflammation-related genes were also strongly upregulated. These
included NFKBID and BCL10, which were involved in the NF-κB
pathway and were upregulated at FDR < 0.05. In addition, the
interferon regulatory factor IRF3 is upregulated at P < 0.05, and IRF3
is co-expressed with AP-1 subunits. The simultaneous upregulation
of AP-1 subunits, NFκB-related genes and interferon regulatory
factors suggested that immune and inflammation responses were
activated in ASD neurons. Upregulated immune response and
neuroinflammation have been consistently observed in ASD
patients by bulk tissue transcriptomic studies largely implicating
glial cells (7, 8). However, our results demonstrated that immune/
neuroinflammatory response was clearly activated in ASD neurons,
and may be mediated by transcription factor AP-1.
We also observed strong downregulation in ASD STG neurons
of GAD1 and GAD2 genes, involved in the biosynthesis of the
inhibitory neurotransmitter GABA. In contrast, CAMK2A and
CAMK2B genes, which are essential for aspects of plasticity at
glutamatergic excitatory synapses, are upregulated at nominal
significance. In addition, co-expressed gene modules that were
upregulated in ASD neurons were mainly enriched in excitatory
neurons, while the downregulated module was primarily enriched
in inhibitory neurons. These data supported the hypothesis that
ASD reflects imbalance of E/I in neuronal communication, also
reported in several brain regions in ASD (19, 20, 39). This is a
report providing molecular-level evidence for imbalance of E/I in
neuronal communication specifically in STG neurons in ASD.
Future studies will focus on the role of snoRNAs in ASD neurons,
and other long and small modulatory non-coding RNAs. Given the
emerging role of snoRNAs as alternative splicing regulators (50,
51), we hypothesize that a coordinated downregulation of multiple
snoRNAs correlates with elevated dysregulation of local splicing
events in ASD neurons. Our data provide evidence supporting this
hypothesis; however, no causal relationship can be determined. It
will be critical to determine if snoRNA dysregulation plays a causal
role in ASD etiology, and if so, pinpoint the underlying mechanism
pnas.org
 and possibly relate to transcript isoforms driving ASD brain and
neuronal phenotype.
In our study, the concordance of ASD-associated transcriptomic
changes in bulk tissue and LCM neurons was relatively low. This
is not surprising given bulk tissue contains various different neu-
ronal and non-neuronal cell types. Neuron-specific changes could
be masked by changes in other cell types when profiling bulk
tissue. The lack of concordance highlighted the necessity of spe-
cifically isolating and profiling each cell type to identify cell-type-
specific transcriptomic signatures.
Age-Associated Differential Expression in STG Points to Altered
Neuronal Activity in ASD. In bulk tissue, genes involved in
GABAergic signaling (GAD1 and GAD2) were upregulated with
age in controls, while downregulated with age in ASD. Multiple
lines of evidence have pointed to deviations in the production,
transmission, and reception of GABAergic inhibitory interneurons,
including decreased numbers of GABAergic interneurons (especially
Parvalbumin neurons) (59) and reduced density of GABA receptors
(60–62). Our findings indicated that the reduction of GAD1 and
GAD2 mRNA levels in ASD brain became more profound with
age, consistent with the observations at cellular level. In addition,
SLC38A1, involved in neurotransmission at glutaminergic and
GABAergic synapses (63), was downregulated in ASD relative to
control STG. SLC38A1 is implicated in Rett Syndrome (64) and
mitochondrial disorders, and its decrease may contribute to the
observed alterations in synapse formation and neural connectivity.
In LCM neurons, the expression of HTRA2 was higher below
age 30 and decreases with age in control neurons, while lower at
younger ages and increasing with age in ASD neurons. HTRA2
is important in maintaining mitochondrial homeostasis (65) and
inducing apoptosis. It is implicated in pathogenesis of neurode-
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.
generation, hypoxic-ischemic damage, and is proposed as a poten-
tial treatment target in neurological diseases (66). Attenuated
HTRA2 activity may lead to neuronal cell death, altered chaperon
activity and autophagy and has been linked to Parkinson’s disease
(67). In addition, increased active form of the OMI/HTRA2 serine
protease has been positively correlated with cholinergic alterations
in AD brain (68). Thus, it is plausible that the altered expression
of HTRA2 with age we observed in ASD brain may be associated
with neuronal alterations during development. These findings
further support the hypothesis of altered neuronal E/I activity,
neuroinflammation, cell death, and mitochondrial dysfunction,
implicated in ASD (69) and suggest treatment windows to target
specific genes to alter their expression trajectories with age.
In summary, our age-related findings support the premise brain
development in individuals with ASD deviates from that of the neu-
rotypical trajectory beginning in childhood and continues to evolve
across the lifespan (70, 71). Although the STG remains relatively
unexplored (72–74), other brain regions display early excess followed
by reductions in volume, connectivity, and cell densities as people
with ASD age through adulthood. Initial excess and overconnectivity
may lead to hyperexcitation, rendering the brain vulnerable to age-
related and pro-inflammatory mechanisms contributing to later
degenerative outcomes. Our findings of altered neuroinflammatory
expression patterns, taken together with reports of excessive micro-
glial activation in STG (74, 75), implicate immune dysfunction in
the pathophysiology of ASD that may exacerbate with age.
Additionally, we found significant relationships with our ASD tran-
scriptome profiles and AD profiles in the same brain region (25),
supporting recent theories of increased susceptibility to neurodegen-
erative and cognitive decline (76, 77). Lastly, our findings of an
age-related decrease in GAD expression in bulk STG tissue, and an
overall downregulation of GAD in neurons, further supports the
PNAS 2023 Vol. 120 No. 10 e2206758120
hypothesis that GABAergic inhibitory neurons are disproportionally
affected in ASD throughout the lifespan.
Mechanistic Modeling Highlighted Pathways and Genes That
Were Directly Associated with ASD in Neurons. We applied two
orthogonal approaches to gain mechanistic insight from our LCM
neuron data. Bayesian network inference built a network from
co-expressed modules and further prioritized hub genes within
selected module(s), identifying BCL10, ELL and GTF2IRD2B as
genes having driving roles in ASD. The approach from Ingenuity
Pathways Analyses first identified upstream regulators that align
with the observed expression changes in our dataset and elucidated
that they share a predicted association with dopamine signaling
pathways. Expansion of the regulatory network to include another
layer of DEGs in our dataset indicated strong overlap and regulatory
connections to many key genes involved in neuronal function and
inflammation (GAD1, GAD2, and many transcriptional regulators).
Interestingly, the expansion of the IPA network further converged on
regulatory molecules BCL10 and RNA polymerase II, of which the
former was directly identified in the independent Bayesian approach,
and the latter associated with ELL. Together these two independent
approaches highlight the role of inflammatory pathways and genes
in ASD etiology.
Limitation and Future Studies
Although our study had a relatively large sample size of 59 human
brains, there are inherent limitations to utilizing postmortem human
brain tissue, including variability in cause of death, co-morbid con-
ditions, medication use, postmortem interval (PMI), etc., Future
studies will have the advantage of more in-depth clinical information
available from newer brain tissue collections such as Autism BrainNet
(SFARI) and the NIH Biobanks to evaluate the relationship of cel-
lular and molecular findings with clinical characteristics. Most of
our subjects were male, consistent with the 4:1 ratio of males/females
with ASD; future studies should include a larger female cohort to
adequately evaluate sex differences. Additionally, the development
of novel tools for profiling the transcriptome such as scRNA-seq
allows for analyses of individual cell types at high resolution; however,
snRNA-seq currently does not provide the depth of coverage to
quantify low-expressed genes and local splicing, especially in human
brain samples that we were able to achieve with LCM neuron sam-
ples. Our study, however, did not distinguish neuronal subtypes, as
we found Histogene the most reliable cell body staining method for
maintaining tissue quality and accurate neuronal identification. Our
future studies will expand on current findings, particularly age-re-
lated changes in GAD implicating GABAergic interneurons, to map
expression in specific cell types in multiple brain regions, in relation
to clinical characteristics, across the human lifespan.
Methods
Bulk Tissue RNA Extraction and Library Preparation. Tissue and clinical data
collection procedures were approved by the institutional review board (IRB) and
Human and Anatomical Specimens Tissue Oversight Committee (HASTOC) at the
University of California, Davis School of Medicine. Informed consent was obtained
from next-of-kin at the time of brain tissue collection for follow-up to collect donor
clinical information to confirm diagnoses by the Autism Tissue Program (now Autism
BrainNet).
Human brain tissue was collected, sectioned coronally and flash-frozen. The
STG was identified anatomically according to “Atlas of the Human Brain” fourth
edition (Maj, Majtanik, Paxinos 2015). Total RNA was extracted using the Direct-zol
RNA MiniPrep (Zymo Research #R2051).
Fifty nanograms of RNA from each sample were used to create strand-specific
total RNA libraries with the NuGEN Ovation Universal RNA-Seq System v2 and
https://doi.org/10.1073/pnas.2206758120 9 of 11
 processed in parallel on the Sciclone NGS automated workstation (Perkin Elmer)
according to manufacturer protocol.
Laser Capture Microdissection, RNA Extraction, and Library Preparation.
Using a Leica LMD-6000 laser capture microdissection system, 100 neurons
from each sample were laser captured directly into lysis buffer. RNA was
extracted using the PicoPure total RNA kit according to manufacturer protocol.
Strand-specific rRNA depleted RNA libraries were prepared from 10 μL of the
final neuronal RNA.
RNA Sequencing, Read Mapping, Quantification of Gene Expression, and
QC. RNA-Seq was performed on Illumina HiSeq4000. Libraries were sequenced to
~50 million 2 × 150 bp reads per sample. Reads were aligned to the GRCH37.p13
(hg19) reference genome using STAR. Gene-level quantifications were calculated
using featureCounts (v1.6.4). Sample outliers were defined as samples with stand-
ardized sample network connectivity Z scores < −2 (78), and were removed. Using
this method, five samples from bulk data, and five samples from neuron data were
removed. Batch effects were accounted for using metrics derived from PicardTools
(v2.21.2).
DGE and Differential Alternative Splicing. DGE analyses were performed
using DESeq2 (1.22.2)(79) with default parameters. Local splicing analysis was
performed using LeafCutter (26) as previously described (10).
Co-expression Network Analysis. Weighted gene co-expression network
analysis (WGCNA) (27) was performed to define modules of co-expressed genes
from RNA-seq data.
Functional Enrichment Analysis. Gene ontology (GO) enrichment was per-
formed using the gProfileR R package (80) and the fGSEA algorithm as imple-
mented in the clusterProfiler R package (81).
Enrichment analyses were also performed using several established, hypoth-
esis-driven gene sets. Significance was determined from permutation-derived
null distribution.
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.
Cell Type Enrichment Analysis. Cell-type enrichment analysis was performed
using the Expression Weighted Cell Type Enrichment (EWCE) package in R (82).
GWAS Enrichment Analysis. Stratified LD score regression (sLDSC) (83) was
used to test whether a gene set of interest is enriched for SNP-heritability in a
given GWAS dataset.
Ingenuity Pathway Analysis. Ingenuity Pathway Analysis (IPA®, QIAGEN) tools
were used to predict the upstream regulators and to build mechanistic networks (84).
Designing and Learning the Bayesian Network Structure. Each of the 18
module eigengenes from our neuron co-expression data serves as one random
variable (node) in our Bayesian network (BN). In addition, we also include diag-
nosis (ASD or CTL) as a binary random variable (node). This design allows us to
1. Y. S. Kim et al., Prevalence of autism spectrum disorders in a total population sample. Am. J.
Psychiatry. 168, 904–912 (2011).
2. M. J. Maenner et al., Prevalence of autism spectrum disorder among children aged 8 years - autism
and developmental disabilities monitoring network, 11 sites, United States, 2016. MMWR Surveill.
Summ. 69, 1–12 (2020).
3. C. Lord et al., Autism spectrum disorder. Nat. Rev. Dis. Primers 6, 5 (2020).
4. S. N. Hansen et al., Recurrence risk of autism in siblings and cousins: A multinational, population-
based study. J. Am. Acad. Child Adolesc. Psychiatry 58, 866–875 (2019).
5. L. Castelbaum, C. M. Sylvester, Y. Zhang, Q. Yu, J. N. Constantino, On the nature of monozygotic twin
concordance and discordance for autistic trait severity: A quantitative analysis. Behav. Genet. 50,
263–272 (2020).
6. T. Gaugler et al., Most genetic risk for autism resides with common variation. Nat. Genet 46,
881–885 (2014).
7. I. Voineagu et al., Transcriptomic analysis of autistic brain reveals convergent molecular pathology.
Nature 474, 380–384 (2011).
8. N. N. Parikshak et al., Genome-wide changes in lncRNA, splicing, and regional gene expression
patterns in autism. Nature 540, 423–427 (2016).
9. M. J. Gandal et al., Shared molecular neuropathology across major psychiatric disorders parallels
polygenic overlap. Science 359, 693–697 (2018).
10. M. J. Gandal et al., Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and
bipolar disorder. Science 362, eaat8127 (2018).
11. S. Gupta et al., Transcriptome analysis reveals dysregulation of innate immune response genes and
neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014).
10 of 11 https://doi.org/10.1073/pnas.2206758120
distinguish between modules that are directly associated with ASD and those
that are indirectly associated with ASD (45).
Prioritizing Genes within Neu-M5. To prioritize genes within the Neu-M5 mod-
ule, we first identified 21 genes within Neu-M5 that are also significant DEGs.
We then built Bayesian network among the 21 genes with method described in
the above section. We selected genes with the highest number of connections as
nodes likely to influence the expression of many other genes within the module.
Alzheimer’s Disease Datasets. Alzheimer's disease DGE data was obtained
from the Mount Sinai/JJ Peters VA MSBB study (25) through Synapse via accession
number syn30821563.
Data, Materials, and Software Availability. All custom code used in this man-
uscript is available at https://github.com/gandallab/ASD_STG_LCM_RNAseq (85).
RNA-seq data from this study is available through the database for Genotypes
and Phenotypes (dbGaP Study Accession ID: phs003208) (86).
ACKNOWLEDGMENTS. We are grateful to the families of our brain donors
for their invaluable gift to autism research. We appreciate the contributions of
Carolyn Komich Hare, clinical Director of Autism BrainNet, for collection of the
ADI-R as well as Robin Riedel, UC Davis, for figure design. Tissue was provided
by the University of Maryland Brain and Tissue Bank, Dr. Daniel Campbell
at Michigan State University, Autism Tissue Program (now Autism BrainNet,
supported by the Simons Foundation), Brain Endowment for Autism Research
Sciences (BEARS) at the UC Davis MIND Institute, and the Harvard Brain Tissue
Resource Center. Laser capture microdissection was conducted using the CAMI
core facility at UC Davis Center for Health and the Environment with instrumen-
tation funding by NIHS10RR-023555 (carried out by A.O.). RNA sequencing
was conducted using the Vincent J. Coates Genomics Sequencing Laboratory
at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.
M.J.G.’s contributions were supported by a SFARI Bridge to Independence
Award, R01-MH121521, R01-MH123922 and IDDRC P50-HD103557 at UCLA.
This research was supported by NIH grants MH108909 (MPI C.M.S. and B.S.),
R01 MH097236 and R21 MH 119650 (C.M.S), and the Intellectual and
Developmental Disabilities Research Center (IDDRC) at the UC Davis MIND
Institute (1U54HD079125).
Author affiliations: aDepartment of Psychiatry and Biobehavioral Sciences, David Geffen
School of Medicine, University of California, Los Angeles, CA 90095; bDepartment of
Human Genetics, David Geffen School of Medicine, University of California, Los Angeles,
CA 90095; cSemel Institute for Neuroscience and Human Behavior, University of
California, Los Angeles, CA 90095; dDepartment of Psychiatry and Behavioral Sciences,
School of Medicine, University of California, Davis, Sacramento, CA 95817; eUniversity
of California, Davis, MIND Institute, Sacramento, CA 95817; fDepartment of Neurology,
University of California, Davis, School of Medicine, Sacramento, CA 95817; gDepartment
of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia,
PA 19104; hDepartment of Genetics, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104; and iLifespan Brain Institute, Penn Med and the
Children’s Hospital of Philadelphia, Philadelphia, PA 19104
12. D. Velmeshev et al., Single-cell genomics identifies cell type-specific molecular changes in autism.
Science 364, 685–689 (2019).
13. M. J. Gandal et al., Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.
Nature 611, 532–539 (2022).
14. R. J. Jou, N. J. Minshew, M. S. Keshavan, M. P. Vitale, A. Y. Hardan, Enlarged right superior
temporal gyrus in children and adolescents with autism. Brain Res. 1360, 205–212
(2010).
15. D. G. Amaral, C. M. Schumann, C. W. Nordahl, Neuroanatomy of autism. Trends Neurosci. 31,
137–145 (2008).
16. D. Wang et al., Comprehensive functional genomic resource and integrative model for the human
brain. Science 362, 8464 (2018).
17. M. E. Feder, G. E. Hofmann, Heat-shock proteins, molecular chaperones, and the stress response:
Evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282 (1999).
18. R. J. Binder, Functions of heat shock proteins in pathways of the innate and adaptive immune
system. J. Immunol. 193, 5765–5771 (2014).
19. V. S. Sohal, J. L. R. Rubenstein, Excitation-inhibition balance as a framework for investigating
mechanisms in neuropsychiatric disorders. Mol. Psychiatry. 24, 1248–1257 (2019).
20. S. B. Nelson, V. Valakh, Excitatory/inhibitory balance and circuit homeostasis in autism spectrum
disorders. Neuron 87, 684–698 (2015).
21. M. L. Chow et al., Age-dependent brain gene expression and copy number anomalies in autism suggest
distinct pathological processes at young versus mature ages. PLoS Genet. 8, e1002592 (2012).
22. B. P. Grone, K. P. Maruska, Three distinct glutamate decarboxylase genes in vertebrates. Sci. Rep. 6,
30507 (2016).
pnas.org
 23. E. Patrick et al., Deconvolving the contributions of cell-type heterogeneity on cortical gene
expression. PLoS Comput. Biol. 16, e1008120 (2020).
24. E. Magnin, Neurodevelopmental and neurodegenerative similarities and interactions: A point of
view about lifelong neurocognitive trajectories. J. Alzheimers Dis. 79, 1397–1407 (2021).
25. M. Wang et al., The Mount Sinai cohort of large-scale genomic, transcriptomic and proteomic data in
Alzheimer’s disease. Sci. Data 5, 1–16 (2018).
26. Y. I. Li et al., Annotation-free quantification of RNA splicing using LeafCutter. Nat. Genetics 50,
151–158 (2018).
27. P. Langfelder, S. Horvath, WGCNA: An R package for weighted correlation network analysis. BMC
Bioinformatics 9, 559 (2008).
28. H. J. Yost, S. Lindquist, RNA splicing is interrupted by heat shock and is rescued by heat shock
protein synthesis. Cell 45, 185–193 (1986).
29. F. K. Satterstrom et al., Large-scale exome sequencing study implicates both developmental and
functional changes in the neurobiology of autism. Cell 180, 568–84.e23 (2020).
30. B. S. Abrahams et al., SFARI Gene 2.0: A community-driven knowledgebase for the autism spectrum
disorders (ASDs). Mol. Autism 4, 36 (2013).
31. K. E. Samocha et al., A framework for the interpretation of de novo mutation in human disease. Nat.
Genet 46, 944–950 (2014).
32. K. J. Karczewski et al., The ExAC browser: Displaying reference data information from over 60 000
exomes. Nucleic Acids Res. 45, D840–D855 (2017).
33. B. Wilkinson et al., The autism-associated gene chromodomain helicase DNA-binding protein 8
(CHD8) regulates noncoding RNAs and autism-related genes. Transl. Psychiatry. 5, e568 (2015).
34. J. Grove et al., Identification of common genetic risk variants for autism spectrum disorder. Nat.
Genet 51, 431–444 (2019).
35. J. Hess, P. Angel, M. Schorpp-Kistner, AP-1 subunits: Quarrel and harmony among siblings. J. Cell
Sci. 117, 5965–5973 (2004).
36. E. C. Warburton et al., Cholinergic neurotransmission is essential for perirhinal cortical plasticity and
recognition memory. Neuron 38, 987–996 (2003).
37. A. Binda et al., A novel KCNJ2 mutation identified in an autistic proband affects the single channel
properties of Kir2.1. Front. Cell Neurosci. 12, 76 (2018).
38. E. Ambrosini et al., Genetically induced dysfunctions of Kir2.1 channels: Implications for short QT3
syndrome and autism-epilepsy phenotype. Hum. Mol. Genet. 23, 4875–4886 (2014).
39. J. L. R. Rubenstein, M. M. Merzenich, Model of autism: Increased ratio of excitation/inhibition in key
neural systems. Genes Brain Behav. 2, 255–267 (2003).
40. F. Renoux et al., The AP1 transcription factor Fosl2 promotes systemic autoimmunity and
inflammation by repressing treg development. Cell Rep. 31, 107826 (2020).
41. S. E. Sweeney, T. B. Kimbler, G. S. Firestein, Synoviocyte innate immune responses: II. Pivotal role of
IFN regulatory factor 3. J. Immunol. 184, 7162–7168 (2010).
42. M. Iwanaszko, M. Kimmel, NF-κB and IRF pathways: Cross-regulation on target genes promoter
level. BMC Genomics 16, 307 (2015).
43. N. Friedman, M. Linial, I. Nachman, D. Pe’er, Using Bayesian networks to analyze expression data. J.
Comput. Boil. 7, 601–620 (2000).
44. B. Zhang et al., Integrated systems approach identifies genetic nodes and networks in late-onset
Alzheimer’s disease. Cell 153, 707–720 (2013).
Downloaded from https://www.pnas.org by 177.159.17.251 on May 3, 2024 from IP address 177.159.17.251.
45. S. Mostafavi et al., A molecular network of the aging human brain provides insights into the
pathology and cognitive decline of Alzheimer’s disease. Nat. Neurosci. 21, 811–819 (2018).
46. T. Kiss, Small nucleolar RNAs: An abundant group of noncoding RNAs with diverse cellular functions.
Cell 109, 145–148 (2002).
47. T. Bratkovič, J. Božič, B. Rogelj, Functional diversity of small nucleolar RNAs. Nucleic Acids Res. 48,
1627–1651 (2020).
48. H. Dvinge, J. Guenthoer, P. L. Porter, R. K. Bradley, RNA components of the spliceosome regulate
tissue- and cancer-specific alternative splicing. Genome Res. 29, 1591–1604 (2019).
49. S. Valadkhan, L. S. Gunawardane, Role of small nuclear RNAs in eukaryotic gene expression. Essays
Biochem. 54, 79–90 (2013).
50. S. Kishore, S. Stamm, The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor
2C. Science 311, 230–232 (2006).
51. M. Falaleeva et al., Dual function of C/D box small nucleolar RNAs in rRNA modification and
alternative pre-mRNA splicing. Proc. Natl. Acad. Sci. U.S.A. 113, E1625–E1634 (2016).
52. A. D’Aniello et al., Amino acids and transaminases activity in ventricular CSF and in brain of normal
and Alzheimer patients. Neurosci. Lett. 388, 49–53 (2005).
53. B. S. Stamova et al., Evidence for differential alternative splicing in blood of young boys with autism
spectrum disorders. Mol. Autism 4, 1–16 (2013).
54. Y. Araki et al., SynGAP isoforms differentially regulate synaptic plasticity and dendritic development.
Elife 9, e56273 (2020).
55. D. Comoletti et al., The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in
protein processing. J. Neurosci. 24, 4889–4893 (2004).
56. N. T. Sprenkle, S. G. Sims, C. L. Sánchez, G. P. Meares, Endoplasmic reticulum stress and
inflammation in the central nervous system. Mol. Neurodegener. 12, 42 (2017).
PNAS 2023 Vol. 120 No. 10 e2206758120
57. A. Crider, A. O. Ahmed, A. Pillai, Altered expression of endoplasmic reticulum stress-related genes in
the middle frontal cortex of subjects with autism spectrum disorder. Mol. Neuropsychiatry 3, 85–91
(2017).
58. M. I. Timberlake, Y. Dwivedi, Linking unfolded protein response to inflammation and depression:
Potential pathologic and therapeutic implications. Mol Psychiatry 24, 987–994 (2019).
59. E. Hashemi, J. Ariza, H. Rogers, S. C. Noctor, V. Martínez-Cerdeño, The number of parvalbumin-
expressing interneurons is decreased in the prefrontal cortex in autism. Cerebral Cortex 27,
1931–1943 (2017).
60. G. J. Blatt et al., Density and distribution of hippocampal neurotransmitter receptors in autism: An
autoradiographic study. J. Autism Dev. Disorders 31, 537–543 (2001).
61. A. L. Oblak, T. T. Gibbs, G. J. Blatt, Reduced GABAA receptors and benzodiazepine binding sites in the
posterior cingulate cortex and fusiform gyrus in autism. Brain Res. 1380, 218–228 (2011).
62. A. L. Oblak, T. T. Gibbs, G. J. Blatt, Decreased GABAB receptors in the cingulate cortex and fusiform
gyrus in autism. J. Neurochem. 114, 1414–1423 (2010).
63. T. Qureshi et al., The glutamine transporter Slc38a1 regulates GABAergic neurotransmission and
synaptic plasticity. Cereb. Cortex 29, 5166–5179 (2019).
64. L.-W. Jin et al., Dysregulation of glutamine transporter SNAT1 in Rett syndrome microglia: A
mechanism for mitochondrial dysfunction and neurotoxicity. J. Neurosci. 35, 2516–2529 (2015).
65. L. V. Walle, M. Lamkanfi, P. Vandenabeele, The mitochondrial serine protease HtrA2/Omi: An
overview. Cell Death Differ. 15, 453–460 (2008).
66. X. J. Su, L. Huang, Y. Qu, D., Mu Progress in research on the role of Omi/HtrA2 in neurological
diseases. Rev. Neurosci. 30, 279–287 (2019).
67. H. Plun-Favreau et al., The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-
associated kinase PINK1. Nat Cell Biol. 9, 1243–1252 (2007).
68. T. Darreh-Shori et al., Increased active OMI/HTRA2 serine protease displays a positive correlation
with cholinergic alterations in the Alzheimer’s disease brain. Mol. Neurobiol. 56, 4601–4619
(2019).
69. K. Kawada, N. Kuramoto, S. Mimori, Possibility that the onset of autism spectrum disorder is induced
by failure of the glutamine-glutamate cycle. Curr. Mol. Pharmacol. 14, 170–174 (2021).
70. C. Ecker, S. Y. Bookheimer, D. G. Murphy, Neuroimaging in autism spectrum disorder: Brain structure
and function across the lifespan. Lancet Neurol. 14, 1121–1134 (2015).
71. T. A. Avino et al., Neuron numbers increase in the human amygdala from birth to adulthood, but not
in autism. Proc. Natl. Acad. Sci. U.S.A. 115, 3710–3715 (2018).
72. B. P. Ander, N. Barger, B. Stamova, F. R. Sharp, C. M. Schumann, Atypical miRNA expression in
temporal cortex associated with dysregulation of immune, cell cycle, and other pathways in autism
spectrum disorders. Mol. Autism 6, 1–13 (2015).
73. B. Stamova, B. P. Ander, N. Barger, F. R. Sharp, C. M. Schumann, Specific regional and age-related
small noncoding RNA expression patterns within superior temporal gyrus of typical human brains
are less distinct in autism brains. J. Child Neurol. 30, 1930–1946 (2015).
74. K. Garbett et al., Immune transcriptome alterations in the temporal cortex of subjects with autism.
Neurobiol. Dis. 30, 303–311 (2008).
75. A. S. Lee, E. C. Azmitia, P. M. Whitaker-Azmitia, Developmental microglial priming in postmortem
autism spectrum disorder temporal cortex. Brain Behav. Immun. 62, 193–202 (2017).
76. G. Vivanti, S. Tao, K. Lyall, D. L. Robins, L. L. Shea, The prevalence and incidence of early-onset
dementia among adults with autism spectrum disorder. Autism Res. 14, 2189–2199 (2021).
77. B. N. Hand, A. M. Angell, L. Harris, L. A. Carpenter, Prevalence of physical and mental health
conditions in Medicare-enrolled, autistic older adults. Autism 24, 755–764 (2020).
78. M. C. Oldham, P. Langfelder, S. Horvath, Network methods for describing sample relationships in
genomic datasets: Application to Huntington’s disease. BMC Syst. Biol. 6, 63 (2012).
79. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq
data with DESeq2. Genome Biol. 15, 1–21 (2014).
80. U. Raudvere et al., g: Profiler: A web server for functional enrichment analysis and conversions of
gene lists (2019 update). Nucleic Acids Res. 47, W191–W198 (2019).
81. G. Yu, L.-G. Wang, Y. Han, Q.-Y., He, clusterProfiler: An R package for comparing biological themes
among gene clusters. OMICS 16, 284–287 (2012).
82. N. G. Skene, S. G. Grant, Identification of vulnerable cell types in major brain disorders using
single cell transcriptomes and expression weighted cell type enrichment. Front Neurosci. 10, 16
(2016).
83. H. K. Finucane et al., Partitioning heritability by functional annotation using genome-wide
association summary statistics. Nat. Genet. 47, 1228–1235 (2015).
84. A. Krämer, J. Green, J. Pollard Jr., S. Tugendreich, Causal analysis approaches in ingenuity pathway
analysis. Bioinformatics 30, 523–530 (2014).
85. P. Zhang, M. J. Gandal, ASD_STG_LCM_RNAseq_paper_code. Github. https://github.com/
gandallab/ASD_STG_LCM_RNAseq. Deposited 27 January 2023.
86. A. Omanska, B. Stamova, C. M. Schumann, B. P. Ander, Transcriptomic profiling of bulk tissue and
laser-capture micro dissected neurons of postmortem human brains in the superior temporal gyrus.
NCBI’s Database of Genotypes and Phenotypes (dbGaP). https://www.ncbi.nlm.nih.gov/projects/gap/
cgi-bin/study.cgi?study_id=phs003208.v1.p1 (Deposited 17 February 2023).
https://doi.org/10.1073/pnas.2206758120 11 of 11