Abstract
Autism spectrum disorder (ASD) is highly heritable but genetically heterogeneous. The affected neural circuits and cell types remain unclear and may vary at different developmental stages. By analyzing multiple sets of human single cell transcriptome profiles, we found that ASD candidates showed enriched gene expression in neurons, especially in inhibitory neurons. ASD candidates were also more likely to be the hubs of the co-expressed module that is highly expressed in inhibitory neurons, a feature not detected for excitatory neurons. In addition, we found that upregulated genes in multiple ASD cortex samples were also enriched with genes highly expressed in inhibitory neurons, suggesting a potential increase of inhibitory neurons and an imbalance in the ratio between excitatory and inhibitory neurons. Furthermore, the downstream targets of several ASD candidates, such as CHD8, EHMT1 and SATB2, also displayed enriched expression in inhibitory neurons. Taken together, our analysis of single cell transcriptomic data suggest that inhibitory neurons may be the major neuron subtype affected by the disruption of ASD gene networks, providing single cell functional evidence to support the excitatory/inhibitory (E/I) imbalance hypothesis.
Introduction
ASD is a class of neurodevelopmental disorders characterized by persistent deficits in social communication/interaction and restricted, repetitive patterns of behaviors, interests or activities (DSM-5) 1. Recent epidemiology studies have reported that 1 in 68 children is diagnosed with ASD, with a 3 to 4-fold increased risk for boys 2, 3. Family and twin studies have found that ASD is highly heritable 4, 5, but the genetic risk factors for ASD are highly heterogeneous and up to one thousand genes are estimated to be involved, with no single gene accounting for >1–2% of the cases 6. These ASD candidate genes converge on several molecular and cellular pathways, such as synaptic function, Wnt-signal and chromatin remodeling 7–12, indicating that ASD pathogenesis is a complicated multidimensional process modulated by genetic factors that play key roles in response to intrinsic developmental signaling and environmental perturbations.
At the cellular level, a human brain can be divided into distinct functional regions that are composed of diverse but densely connected cell types. It has been reported that ASD risk genes form co-expression networks that are expressed at relatively higher levels in specific embryonic prefrontal cortex regions and layers 13, 14, and ASD mutations could potentially affect certain brain areas and cell types more strongly than others 15. For example, Xu et al. previously developed a method (“cell type-specific expression analysis”) to analyze microarray gene expression data from mouse and human brains, including cell type data from translating ribosome affinity purification (TRAP) technology, and found that multiple cell types could be implicated in ASD 16, e.g., astrocytes, glia and cortical interneurons. Subsequently, Zhang et al. also used TRAP data from mouse lines and observed that an expression signature shared by ASD risk genes is a strong and positive association with specific neurons in different brain regions, including cortical neurons 17.
A limitation of these previous studies is related to the concern that the resolution of cell types may not be sufficient, in addition to other limitations specifically related to the microarray platform. This can be addressed by single cell RNA-seq (scRNA-seq) analysis that measures gene expression profiles for hundreds to thousands of cells in a tissue sample simultaneously, which can resolve cell types and reveal expression heterogeneity 18. With a mouse scRNA-seq dataset 19 and a novel computational method, Skene et al. suggested that genetic susceptibility of ASD primarily affected interneurons and pyramidal neurons 20. The method is called “expression weighted cell-type enrichment” (EWCE), which evaluates statistically whether a set of genes shows higher expression in a particular cell type than what is expected by chance 20.
While the above studies have suggested that ASD risk genes can have cell type specific functions and expression patterns, and some brain cell types may be more prone to the effects of ASD-associated mutations, no similar studies have been performed using human cell type-specific functional genomic data, such as scRNA-seq data. This is important because it has been shown that some gene expression modules are human specific, several of which are correlated with brain disorders, such as Alzheimer’s disease 21, despite the extensive global network similarity of the human and mouse brain transcriptomes. Our previous study of the transcriptional regulatory network modulated by a neural master regulator, REST/NRSF, also showed that ASD genes are enriched among human specific REST targets 22. Moreover, the human brain is much more complex than the mouse brain, especially in some regions, such as the frontal and temporal lobes, which have undergone enormous changes during primate evolution 23. In addition, no systematic studies related to ASD have been carried out in which excitatory and inhibitory neuronal transcriptomes have been compared, despite the long-standing E/I imbalance hypothesis, which has been proposed as a model to explain some ASD-related behaviors 24-27. Therefore, to address if some cell types are more prone to genetic network disruptions potentially occurring in the brains of individuals with ASD, we have collected multiple human neural or brain expression datasets, most of which were derived from advanced scRNA-seq analysis, and evaluated if genes implicated in ASD show different expression profiles across human neural cell types. The gene sets in our study include a) ASD candidate genes, b) differentially expressed genes between ASD individuals and controls, and c) downstream targets of ASD candidates. We found that these genes consistently show significantly enriched expression in human neurons, particularly inhibitory neuron, suggesting that inhibitory neuron is the major cell type affected in ASD. This finding is consistent with the hypothesis that a disruption of the balance between inhibitory and excitatory signaling could be an important underlying mechanism of ASD pathogenesis.
Materials and Methods
Human single cell RNA-seq data
Four sets of human scRNA-seq data were analyzed. For the fetal brain and cerebral organoid datasets 41 and the adult brain dataset 42, raw scRNA-seq reads were aligned to the human reference genome (GRCh37/hg19) using STAR (ver. 2.0.13) 43. Duplicate reads were removed using Samtools (ver. 0.1.19) 44, 45. Gene length and uniquely mapped reads for each gene were calculated using featureCounts in subread package (ver. 1.4.6) 46 with gene models from Ensembl release 74. Fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated using R (https://www.r-project.org/) according to its definition. For the neuron subtype dataset for excitatory and inhibitory neurons, transcripts per kilobase million (TPMs) were obtained from the original paper 47. For each cell type, mean of log2(FPKM or TPM) across all samples were calculated and imported into the EWCE 20 to determine enriched expression. In all four cases, the authors’ classification of cell types was used.
Lists of genes associated with ASD, schizophrenia and other brain disorders
ASD candidate genes were downloaded from the SFARI database (https://gene.sfari.org/autdb/GS_Home.do; genes scored as high confidence, to minimal evidence and syndromic) and the AutismKB (core dataset) 28. The two schizophrenia gene lists were from the SZgene database 29 and a recent GWAS report 30. Bipolar disorder associated genes were from the BDgene database 31. Other gene lists associated with brain diseases were described in our previous publication 22. Genes encoding excitatory and inhibitory postsynaptic density (PSD) proteins were from a previous study by Uezu et al 32. The genes associated with human height were from a previous GWAS 33.
Differentially expressed genes between ASD and controls
Gene expression in postmortem cortices (“Cortex1”) 34 was used to detect differentially expressed genes by GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/), which is defined here as FDR < 0.05 and fold change >1.3 - the same criteria as used in the original paper. Differentially expressed genes in blood 35 were also detected by GEO2R and defined as p<0.05. Gene lists from other brain-related samples, including postmortem cortices (“Cortex2”36 and “Cortex3”37), induced pluripotent stem cell (iPSC)-derived cerebral organoids (“Organoid”)38, neural progenitor cells (NPC)39, and neurons (“Neuronl” 39 and “Neuron2” 40), were obtained from the original papers.
Downstream genes of ASD candidates
CHD8-regulated genes in NPCs, neurons 50 and cerebral organoids 51, CYFIP1-regulated genes 52, TCF4 and EHMT1-regulated genes 53, MBD5 and SATB2-regulated genes 54, NRXN1-regulated genes 55 and ZNF804A-regulated genes 56 were from studies where the expression of a known ASD candidate was reduced by knockout or knockdown. Gene lists were obtained from the original papers.
Weighted gene co-expression network analysis (WGCNA)
Signed co-expression networks were built using the WGCNA package 48. The power of 18 was chosen, and blockwiseModules function was performed to build networks. Logistic regression was used to find modules expressed higher in excitatory or inhibitory neurons using eigengenes. P values were corrected by multiple testing to generate FDR. ToppGene 49 was used to find Gene Ontology categories enriched in modules.
Results
ASD candidate genes show enriched expression in neurons, especially inhibitory neurons
It has generally been supposed that functional disruptions of a gene more likely affect the cells or tissues where the gene is highly expressed. Such a principle has often been used to support the discovery of risk genes from genetic studies in schizophrenia and ASD 30, 57. Accordingly, we have used the EWCE method to test what brain cell types are more likely to be affected by genes implicated in ASD, using transcriptomic data containing cell type identifies. Throughout this paper, the term “enrichment expression” in a particular cell type refers to a set of genes that have a higher level of expression within this cell type than expected by chance, as described in the EWCE method 20. The method also accounts for a gene’s overall expression across all cell types in a comparison. We started with scRNA-seq expression data from six cell types from adult human brains (21-63 years old; a total of 285 cells), including neurons, microglia, and astrocytes (Figure 1A) 42. First, as a negative control, we found that genes associated with human height 33 showed no enrichment of expression in any of the six cell types in test (Figure 1). Conversely, as a positive control, genes encoding postsynaptic density proteins (PSD) proteins showed significant enrichment in neuron expression (Figure 1).
Our analysis of the ASD candidates, obtained from either the SFARI (https://gene.sfari.org/autdb/GS_Home.do) or the AutismKB 28, demonstrated that their expression was significantly enriched in human adult neurons and oligodendrocyte precursor cells (OPC) but not astrocytes and microglia (Figure 1A). As ASD is an early developmental disorder, we repeated the same analysis using a single cell transcriptome dataset from human fetal brains, including 226 single-cell transcriptomes from 12- and 13-wk post-conception neocortex specimens 41. The cell types in the fetal brain were classified differently from those in adult brains. We found that in comparison to apical and basal progenitors, ASD candidates were significantly enriched in neurons, especially mature neurons (“N2” and “N3”) in fetal brains (Figure 1B). We also found schizophrenia and bipolar disorder associated genes were similarly enriched in mature neurons (Figure 1B), consist with the known overlap of genetic risk factors among these disorders 58.
Meanwhile, genes associated with several other brain diseases, such as Alzheimer and Huntington, showed no significantly enriched expression in any of these cell types (Figure 1). Next, to study whether ASD candidates are enriched in neurons in specific brain regions, we analyzed single cell transcriptome data of cerebral organoids, including 495 single-cell transcriptomes 41. Again, compared with NPCs, ASD candidates displayed significantly enriched expression in neurons - both dorsal and ventral forebrain neurons, as were schizophrenia and bipolar disease associated genes (Figure 1C). While not quite surprising, our analysis of these three cell type transcriptomic datasets showed that neurons, both early fetal neurons and adult neurons, are a major cell type affected by ASD mutations, probably more so than neural progenitors. Finally, neurons could be largely classified into two major subtypes: excitatory and inhibitory neurons. Using scRNA-seq data of neuronal subtypes, including 3,083 single-cell transcriptomes from six cortical regions of a control normal 51-year-old female postmortem brain 47, we found that the expression of ASD candidates was significantly enriched in inhibitory neurons, especially among the subtypes “In1” and “1n3” (Figure 1D), which are superficial layer inhibitory neurons that originate from lateral ganglionic eminences 47. These results suggest that functional disruptions of ASD genes as a group can affect inhibitory neurons more than excitatory neurons. Although it remains to be established with functional assays, this finding indicates that inhibitory neuron transcriptome dysregulation can occur in ASD brains, which is consistent with the E/I imbalance hypothesis in ASD 24, 59–62. GABAergic neurotransmission appears to play a role in both schizophrenia and bipolar disorders as well 63, 64. However, our results suggest that bipolar disorder but not schizophrenia-associated genes were significantly enriched among highly expressing genes in inhibitory neurons.
ASD candidate genes are more likely to be hubs of co-expression modules in inhibitory neurons
To further study the roles of ASD candidate genes in inhibitory neurons, we performed WGCNA to build a co-expression network from the neural subtype transcriptome data 47 (Fig S1A), resulting in 73 modules (Fig S1B). One of them showed high expression in excitatory neurons and contained 1,936 genes that were enriched for functions related to synaptic signaling, neuron projection and morphogenesis, as well as genes expressed in excitatory synapses (Fig S1C). A different module contained 951 genes that were highly expressed in inhibitory neurons. They were enriched with genes involved in neurogenesis, positive regulation of synaptic transmission, and the GABA shunt (Fig S1D). Consistent with the EWCE result, ASD candidates, from both the SFARI and AutismKB, were more significantly enriched in the module highly expressed in inhibitory neurons (OR = 2.38, p = 5.94e-07, Fisher’s exact test, one-tailed) than the module highly expressed in excitatory neurons (OR = 1.42, p = 0.018, Fisher’s exact test, one-tailed). Among the hub genes in the inhibitory module, nine were ASD candidates (Fig 2A), including three genes encoding transcription factors (ARX, DLX2 and DLX6) that are important for appropriate migration of inhibitory neurons to the cortex 65, and three genes (SLC6A1, GAD1, ALDH5A1) that participate in GABA synthesis, release, reuptake and degradation, as described in the Reactome pathway 66. Notably, those ASD candidate genes had more connections in the inhibitory module than non-ASD candidates (p = 0.0057, Wilcoxon test; Fig 2B), suggesting that ASD candidates tend to be the hubs in inhibitory module, and consequently, disease-associated mutations would likely lead to a disruption of the co-expression network. By comparison, in the excitatory module, ASD and non-ASD candidate genes had similar connections (p = 0.72, Wilcoxon test; Fig 2C, 2D).
Genes up-regulated in ASD-derived neuronal samples show enrichment in inhibitory neurons
Because of the extensive genetic heterogeneity in ASD, investigators have carried out transcriptomic studies in postmortem samples or ASD patient-derived neural samples with the goals of finding common pathways and cellular processes dysregulated in ASD brains or neural samples 34-40. We thus decided to study whether differentially expressed genes (DEGs) in molecular studies carried out between ASD and control subjects exhibited similar cell type-biased expression patterns as ASD candidate genes identified from genetic studies. We obtained DEGs in ASD brain or blood samples and analyzed their expression across brain cell types. Since, as shown above, we uncovered the biased expression pattern of ASD candidate genes (from SFARI or AutismKB), they were excluded for this analysis, in order to focus on the downstream effects. In ASD cortex samples, up-regulated genes were enriched with genes highly expressed in adult astrocytes and microglia (Fig S2A), whereas down-regulated genes were enriched with genes highly expressed in neurons (Fig S2B). This is consistent with previous reports 34, 36, 37, but extends the finding to relatively mature neurons and both dorsal and ventral forebrain neurons (Fig S2B). We also found up-regulated genes in ASD cortex samples were enriched for highly expressed genes in NPCs (Fig S2A), a pattern not detected when ASD candidates were analyzed (Figure 1). However, genes up-regulated in NPCs, neurons and cerebral organoids derived from ASD iPSC-lines showed enriched expression in neurons (Fig S2A), while down-regulated genes in the patient-derived samples were enriched with genes expressed highly in astrocytes, microglia and NPCs (Fig S2B). These results suggest that cell types can be affected differently in early and late developing ASD brains. The difference may also reflect primary vs secondary effects. However, in our comparison of excitatory vs inhibitory neurons, we found that up-regulated genes in both postmortem cortices and cerebral organoids were similarly enriched with genes highly expressed in inhibitory neurons (Fig 3A). The down-regulated genes from cortices and iPSC-derived neurons or cerebral organoids exhibited opposite enrichments, with the former enriched for high expression in excitatory and the latter in inhibitory neurons (Fig 3B). Importantly, dysregulated genes in ASD blood samples 35 did not exhibit any significant pattern of expression enrichment.
Downstream transcriptional targets of key ASD candidates are enriched among genes expressed highly in inhibitory neurons
Finally, we studied whether the downstream targets of ASD candidates genes show different expression enrichment patterns between inhibitory and excitatory neurons by analyzing the DEGs in human neural samples in which the expression of several top ASD (or schizophrenia) candidate genes have been reduced by either knockout or knockdown. We found that CHD8, EHMT1 and SATB2 regulated genes were exclusively enriched in inhibitory neurons (Figure 4). Moreover, a general enrichment in inhibitory neuronal genes, especially those in “In1/2/3” classes, was found among the targets of ASD candidates (Figure 4). Among the downstream targets, DLX1, a transcription factor critical for inhibitory neuron function is markedly upregulated in ASD patient-derived telencephalic organoids 38 and CHD8 knockout cerebral organoids 51, but GAD1, an inhibitory neuron marker, was downregulated in SATB2 knockdown samples 54. We analyzed DEGs from CYFIP1 knockdown in NPCs derived from three independent iPSC-lines and found both common and distinct enriched expression patterns. DEGs from two lines (C2 and C5) were enriched in inhibitory neurons, but C4 DEGs showed enriched expression in excitatory neurons (Figure 4). This difference could reflect the limited overlap of the DEGs 52, but also suggests the intriguing possibility that E/I imbalances are affected by inter-individual differences in genetic background. We should point out that CHD8 and EHMT1 are expressed at a similar level in excitatory and inhibitory neurons, but SATB2 is expressed at a higher level in excitatory neurons. These findings further suggest that some ASD genes can affect the expression of key genes important for inhibitory and excitatory neurons and their targets may be involved in the interaction or signaling balance between the two types of neurons.
Discussion
By integrating ASD candidates, dysregulated genes in ASD samples and downstream targets of ASD candidates with recently published human scRNA-seq datasets, we found that ASD-associated genes exhibited enriched expression in neurons, especially inhibitory neurons, with some developmental stage differences. The enrichment of inhibitory neuronal expression among ASD candidate genes provides molecular support for the finding that deficits in inhibitory neuronal function occurs in some syndromes with autism-associated behaviors, such as individuals with ARX mutations 67, 68, Dravet syndrome caused by loss-of-function (LoF) mutations in SCN1A 69, and Tuberous Sclerosis caused by mutations in TSC1/2 70, 71 (for review, see 72). Our current findings are in line with the long-standing hypothesis that E/I signaling imbalance contributes to ASD. The attractive theory of an increase in the ratio between excitatory and inhibitory signaling provides a plausible explanation for the relative reduction in GABAergic signaling found in patients with ASD and their propensity to develop epilepsy 72. However, a relative excess of inhibitory neuronal activity has been observed in mouse models of Rett Syndrome 73, and mice with a targeted Mecp2 deletion restricted to GABAergic inhibitory neurons recapitulates most of the ASD-like features observed in animal models 74, while restoring Mecp2 expression reverses some of the phenotypical defects 75, 76.
Our analysis showed enriched expression in inhibitory neurons for upregulated but not down-regulated genes in ASD samples. This seems inconsistent with the enriched expression of ASD candidates in inhibitory neurons, assuming their mutations lead to reduced expression and functional loss. One possibility is that some ASD candidates may function as transcriptional inhibitors or the abnormal expression of some ASD candidates could lead to an increase in the number of inhibitory neurons, in a subset of ASD subjects or in certain brain regions, perhaps as a compensation mechanism for a reduction of GABA receptors (or GABAergic function) in individual inhibitory neurons 59. However, previous studies have reported an overproduction of GABAergic inhibitory neurons in ASD iPSC-derived organoids 38 and neural cells 39, with the former likely resulting from increased FOXG1 expression 38, suggesting that an increase in inhibitory interneuron function could be due to a direct effect of some candidate genes. Another key transcription factor in GABAergic interneuron differentiation, DLX1, was also upregulated in CHD8 knockout NPCs, neurons,50 and cerebral organoids 51. Furthermore, our study indicates that both primary and secondary ASD-affected genes may play roles in inhibitory neurogenesis and function, contributing to ASD pathogenesis. We should note that when, where and how an E/I imbalance contributes to ASD is unclear and certainly beyond the scope of the current study. Nevertheless, it is conceivable that E/I imbalance may tilt to one direction in a subset of ASD but to the other in a different subset.
Since neuronal subtype transcriptomes used in the current study were from an adult female brain 47, and there are significant transcriptional (and structural) differences in the brain between the pre- to post-natal period, and from the teenage to adult stage 77, it would be interesting to perform a similar EWCE study using scRNA-seq data from prenatal or fetal neurons in multiple brain regions from both sexes. Considering our findings, it is interesting to note that drugs targeting inhibitory neuron function are being developed to treat ASD78, Consequently, it would be valuable to study their effects in early and late developing brains, animal models, iPSC models, and in ASD subjects using brain imaging and electrophysiology to fully explore the therapeutic potential of such drugs.
Finally, we found that upregulated genes in postmortem ASD brains were enriched in microglia and astrocytes, which is consistent with original reports based on the mouse transcriptome 34, 36. This is consistent with the findings that activated microglia and astrocytosis occur in multiple brain regions of ASD patients 79, 80. However, ASD candidate themselves did not show such an enrichment in our analysis. Thus, dysregulation of neuron-glia signaling might be a secondary process in response to the initial insults elicited by the primary casual genetic variants, a testable hypothesis.
Conflict of Interest
The authors do not have any financial disclosures or other conflicts of interest to declare.
Acknowledgements
This study is supported by NIH grants (MH099427 to HL and HL133120 to DZ). We thank the High Performance Computing of Albert Einstein College of Medicine for computing support.