Transcriptomic analysis reveals increased immune response and oligodendrocyte development in somatosensory cortex
To investigate the effect of Pten heterozygous loss on somatosensory cortex (SSC) at different ages, the activation state of PI3K/mTOR signaling pathway was first characterized. Postnatal day 30 and 42 (P30 and P42) were selected as higher spine density and autistic phenotypes were observed in these stages in Tsc2 null mice, respectively.(43) We found that there were observable increases of p-S6/S6 levels amongst all groups in Pten+/− mice versus littermate control although not statistically significant (Additional file 1: Fig. S1A-B). This suggests subtle cell type- or regional-specific dysregulation in p-S6 in SSC.
To delineate the molecular changes in SSC of Pten+/− mice, RNA-Seq data were analyzed with DESeq2 software package. Irrespective of age and gender, there were an overall 301 upregulated and 50 downregulated DEGs in the Pten+/− group compared to their littermate controls (Fig. 1A). Amongst the top 10 most significant DEGs, 8 of them were upregulated (Fig. 1A). ADGRG1, LGI3 and NEFM are known to play a role in neural development and are associated with neurological diseases, such as epilepsy (Additional file 1: Fig. S1C). Additional upregulated genes such as C1QC and CSF1R are known immune related genes.(44, 45) Conversely, RPRM was downregulated and is involved in p53-dependent G2 arrest of the cell cycle.(46) Furthermore, relatively more DEGs were observed in the following two groups – female irrespectively of age, and P42 irrespective of gender (Fig. 1A).
Gene ontology (GO) and canonical pathways enrichment analysis revealed that immune response and vasculature development were amongst the top upregulated pathways in P30 male (P30M) (Fig. 1B; additional file 1: S1D-E). Similarly enriched terms, like artery morphogenesis, cytokine production and leukocyte migration, were upregulated in P30 female (P30F) (Fig. 1B). Immune related term, such as dendritic cell differentiation, could also be found in P42M. In P42M and P42F, ensheathment of neurons was a shared enriched term (Fig. 1B). Downregulated terms and pathways related to ribosome, translation of protein and protein targeting can only be found in P30M (Fig. 1C; additional file 1: S1F). To compare the enriched GO terms amongst groups, more common GO terms were shared between P30M and P30F (Additional file 1: Fig. S1D), most of which were related to inflammatory/immune response and regulation of PI3K signaling, while terms related to oligodendrocyte were shared amongst all groups except P42F (Additional file 1: Fig. S1D). Taken together, immune response appears to be commonly enriched in P30 groups, while oligodendrocyte differentiation is a common transcriptomic change amongst all groups.
As PTEN has been extensively implicated in ASD, we compared the DEGs of our mice to the weighted gene co-expression network of human brain development.(42) We found that module 8, 13, 15 and 18 were significantly enriched (Additional file 1: Fig. S1G), and they were negative regulation of neuron differentiation, synaptic transmission, response to virus and defense response, respectively (Additional file 1: Fig. S1H).(42) Amongst neuron/synaptic transmission genes and virus/defense response genes, 9 genes were shared by these modules, LGALS3BP, SLC1A3, CYBRD1, TLN1, LPAR1, ATP1A2, SLCO2B1, CLDN11 and B2M (Additional file 2: Table S1). Only module 17 was enriched with downregulated DEGs. This module was also related to synaptic transmission, with 12 shared DEGs, TBRG1, GPRASP1, BEX4, SSTR1, YPEL4, ARPP21, SNCA, CDH9, OPRK1, NCALD, ZNF365 and ABI3BP. Of note, similar transcriptional alterations have been observed in the Ptenm3m4 mutant mouse model.(47)
Proteomic analysis of somatosensory cortex reveals perturbation of dendritic spine development, keratinization and hamartoma signatures
Proteomics analysis was carried out using SSC of the same mice used for RNA-Seq analysis. P30M group was chosen since significant increase in spine density was observed in Tsc2 null mice with mTOR hyperactivation.(43) Amongst proteins with human orthologs, 545 proteins were found with FDR value (Benjamini & Hochberg-corrected) of < 0.05 and consisted of 248 upregulated and 297 downregulated proteins (Fig. 2A). By integrating DEPs and DEGs of all pooled groups, only 15 were upregulated and 4 were downregulated consistently (Fig. 2D; additional file 1: Fig. S2A). Intriguingly, by inspecting them in Brain RNA-Seq,(48, 49) we observed three groups of upregulated proteins. One mainly expressed in microglia/macrophage, including HAVCR2, CRYBB1, C1QC, MPEG1 and TLN1. The second group mainly expressed in oligodendrocytes, including GSN, CERS2 and S100A6. The third group expressed widely in different neural cells - GRB14 and PLIN3 expressed in oligodendrocytes and endothelia; CD82 expressed in oligodendrocytes, microglia/macrophage and endothelia; and BCAN expressed in oligodendrocytes and astrocytes (Additional file 1: Fig. S2A). On the contrary, the downregulated proteins, FXYD6 mainly expressed in neurons and oligodendrocyte precursor cell (OPC); and ATP2B4 and GPM6A expressed in astrocytes, neurons and OPC (Additional file 1: Fig. S2A). These results suggest that different neural cells may be affected differently by Pten heterozygous loss.
Next, we observed that GO and canonical pathways enrichment analysis only enriched with downregulated DEP, and postsynaptic specialization organization and dendritic spine development were amongst the top neural related pathways in Pten+/− SSC proteome (Fig. 2B). Interestingly, keratinization, which is one of the clinical diagnostic criteria for Cowden Syndrome, was also perturbed but its function in brain development is unknown. To understand the relationship between the DEP and enriched pathways, we filtered out proteins by FDR over 0.05, then plotted the DEP against the involved pathways (Additional file 1: Fig. S2B). We observed PTEN, RELN, PSEN1, TIAM1 and PPFIA2 shared amongst postsynaptic and dendritic spine development, whereas DSC1, KRT2, KRT15, KRT1 and KRT77 were associated with keratinization and skin development (Additional file 2: Table S2a). NRIP1 inactivation was involved in cognitive impairments in mice.(50) de novo mutations in KAT5(51) and WASF1(52) were associated with cerebral malformations, seizures, and developmental delay. GJA1 is predominantly located at astrocytic GAP junctions, and has been shown to modulate synaptic plasticity and predicted to be a driver of Alzheimer’s disease.(53) Taken together, these enrichments reveal downregulated DEPs in Pten+/− SSC can potentially influence cognitive functions by changing the synaptic development, while keratinization is novel and requires further investigation.
To understand how these DEPs were associated with known phenotypes, we performed enrichment analysis with human phenotype ontology (HPO) (Fig. 2C; additional file 1: Fig. S2C; additional file 2: Table S2b). Benign tumor-like growth is a diagnostic feature of PHTS. Consistently, our DEPs were enriched with gene sets related to aberrant hypertrophic growth. Hamartoma, hamartoma and neoplasm of the eye were associated with KRT1, VHL and SLC25A11. Various skin abnormalities such as, hyperkeratosis, abnormality of the plantar skin of foot, palmoplantar keratoderma, subcutaneous nodule and erythema were enriched for GJA1, KRT1, KRT2, PSEN1 and INSR. Hyperostosis, abnormality of odontoid tissue and carious teeth are problems related to bone or hard tissues, and were characterized with common proteins, such as GJA1 and SLC24A4. Collectively, proteins downregulated in cortex of Pten+/− mouse model shared amongst PHTS pathologies on multiple tissues.
Transcriptomic analysis of Pten haploinsufficient primary neural cells reveal major perturbation of immediate early genes in neuron
To understand the effect of Pten haploinsufficiency on three important neural cell types, we first investigated the PI3K pathway of primary cultures of neuroprogenitor cell (NPC), astrocyte (AST) and cortical neuron (PCN). NPC at E12-13 was chosen because they are neural lineage cells which undergoes multiple cell fate determination. Moreover, The level of PTEN in Pten+/− NPC was reduced by > 70% but the levels of p-AKT and p-S6 were not significantly elevated. (Additional file 1: Fig. S3A-B). These results suggest that the remaining wild-type allele in Pten+/− NPC is sufficient to suppress aberrant PI3K hyperactivation. For primary astrocytes (AST) cultured from frontal cortexes, the level of p-AKT but not p-S6 was significantly increased in Pten+/− concurrently with over 70% reduction of PTEN protein levels (Additional file 1: Fig S3C-D). These results suggest that AKT activity may be more sensitive to PTEN depletion in primary astrocytes. Lastly, for PCN cultured from frontal cortexes of E15.5 to E16.5 mouse embryos, Pten heterozygosity did not alter p-AKT and p-S6, and PTEN level was reduced by half as expected (Additional file 1: Fig. S3E-F). These suggest PI3K signaling is not affected by PTEN depletion in PCN. Collectively, only very subtle or cell-type specific effect on PI3K signaling is observed in Pten+/− neural cells.
Next, we investigated the transcriptomic changes in Pten+/− neural cells compared with littermate control. Overall, NPC was the least affected without any DEGs discovered. AST had 59 upregulated and 38 downregulated genes. Interestingly, PCN had only 5 upregulated but 57 downregulated genes (Fig. 3A). Notably, most of the downregulated DEGs in Pten+/− PCN belonged to immediate-early response genes (IEGs), namely, Btg2, Dusp1, Erg1, Fos, Fosb, Ier2, Jun, Junb, Maff, Nr4a1, Nr4a2, Npas4, Ptp4a1, and Trib1 (GSE190879). IEGs are known to be involved in synaptic plasticity and memory formation, as well as implicated in psychiatric disorders (54–57). It is paradoxical that these IEGs could not be detected as DEGs in Pten+/− SSC (data not shown).
Enrichment analysis for GO gene sets and canonical pathways revealed that NPC had the least number of enriched terms. Only a few cellular component and pathways about extracellular matrix (ECM), platelet granule and cell adhesion were upregulated and circadian clock was the only suppressed pathway (Fig. 3B, D, & E). More enriched GO terms and pathways were observed for AST (Fig. 3B, D & E). Some were similar to NPC, such as upregulated ECM-related and cell adhesion. Unique enrichment included upregulation of blood circulation, muscle contraction, protein kinase and metalloendopeptidase activity, interleukin 2 family signaling and heparin sulfate/heparin (HS-GAG) metabolism, while downregulated pathways were related to meiosis regulation. Finally, PCN had quite uniquely enriched gene sets (Fig. 3B-E). Upregulated gene sets were mostly related to mitochondrial functions and energy production, such as ATP synthesis coupled electron transport, respirasome, sugar metabolism and TCA cycle. Downregulated gene sets included functions such as pri-miRNA transcription by RNA polymerase II, DNA binding transcription activator activity, long term synaptic depression and long-term memory. As a whole, the above data suggest that heterozygous loss of Pten induces undetectable to very mild gene expression and functional perturbations in different neural cells.
Transcriptomic analysis of Pten knockout primary neural cells uncover neural cell-specific signatures
Pten heterozygous loss only resulted in subtle transcriptional changes. We hypothesized that the complete knockout of Pten could unmask disease associated DEGs unambiguously. Nestin (Nes)-Cre transgenic mice were crossed to Ptenfl/fl mice to generate complete knockout (Nes-KO). The brains of Nes-KO embryos were visually distinguishable at E16.5 from littermate controls (Ctrl) and more striking at P0 (Additional file 1: Fig. S4). Thus, PTEN is required for proper brain development at the commencement of neurogenesis.(58) NPC, AST and PCN were cultured as aforementioned (Fig. 4A, D, & G). Western blotting analysis showed that p-AKT was upregulated in all neural cells (Fig. 4B, C, E, F, H, & I). Interestingly, other PTEN-related signaling molecules, such as pS6, p-mTOR and pGSK3β, p-FOXO1, and p-ERK were not significantly altered in NPC (Fig. 4B-C). On the contrary, levels of p-S6 and p-GSK3β were elevated in AST and PCN, which were consistent with previous studies.(59, 60) These data suggest that different neural cell types have distinct responses to PTEN knockout.
Furthermore, the levels of early neuronal marker, NeuN; and astrocyte marker, GFAP, were not changed in Nes-KO NPC (Fig. 4B). This reflects that PTEN loss does not affect neural differentiation of NPCs obtained at E12-13. For AST, we observed elevated expression of GFAP in Nes-KO cells (Fig. 4E). Increased GFAP level may suggest reactive astrogliosis commonly observed in hypertrophic astrocytes.(15, 61) Therefore, the complete knockout of PTEN in AST can lead to the hyperactivation of PI3K pathway and promoting astrogliosis. For PCN, we noted that level of NF68, a major constituent of the axonal cytoskeleton(62, 63), was drastically increased in Nes-KO group when cultures were matured from DIV5 to DIV14 (Fig. 4H). Also, hypertrophic neurons with thickening neurite were observed at DIV14 (Fig. 4G). Thus, PTEN complete depletion in neurons leads to PI3K activation and is associated with hypertrophic axonal growth.
RNA-Seq analysis revealed that Nes-KO NPC had smallest number of DEGs, with 1,070 upregulated and 662 downregulated protein-coding genes but only 170 upregulated and 9 downregulated genes with log2FC > 1 (Fig. 5A). Nes-KO AST had 3,359 upregulated and 2,615 downregulated DEGs with 1,926 upregulated and 1,118 downregulated genes over log2FC of 1. Strikingly, the maximal expression differences were ranging from log2FC -7.8 to + 12.7. Nes-KO PCN had 2,541 upregulated and 2,462 downregulated DEGs with 694 and 439 of those were log2FC > 1. Collectively, the transcriptomes of Nes-KO AST are the most perturbed compared to other neural cells with the complete loss of PTEN.
Gene set enrichment analysis showed that Nes-KO NPC had upregulated energy metabolic processes, such as hexose catabolic, NAD and NADH metabolic processes, carbohydrate binding and gluconeogenesis (Fig. 5B; additional file 1: Fig. S5A), and downregulated ribosome related processes, such as ribosome and ribonucleoprotein complex biogenesis, ribosomal subunit, preribosome, ribosome and spliceosomal complex, along with related molecular functions such as DNA replication and rRNA binding (Fig. 5C; additional file 1: Fig. S5B). These results indicate that upregulated energy metabolism and downregulated mRNA translation and protein expression are signatures of Nes-KO NPC.
In Nes-KO AST, components of cilium and ribosome, as well as mitosis related pathways, were amongst the top upregulated change (Fig. 5B; additional file 1: Fig. S5A), whereas, relatively fewer downregulated functions, such as the insulin-like growth factor-1 receptor (IGF-1R) signaling pathway, endothelial cell migration, growth factor activity and JAK-STAT signaling pathway, were discovered (Fig. 5C; additional file 1: Fig. S5B). These results point to upregulated activities of cilium, mRNA translation as well as mitosis are top signatures of Nes-KO AST.
In Nes-KO PCN, major upregulated changes include extracellular matrix components (ECM) and cell adhesion, sterol metabolic process, leukocyte transendothelial migration and MET activates PTK2 signaling (Fig. 5B; additional file 1: Fig. S5A) but downregulated perturbations mainly involved forebrain neuron generation and differentiation (Fig. 5C; additional file 1: Fig. S5B). Of note, most of these downregulated genes are confined to GABAergic neurons or act as determinant of differentiation into inhibitory neurons, such as Ascl1,(64) Lhx6, Arx, Dlx1, Dlx2, Dlx5,(65) Erbb4,(66) Prox1(67) and Foxg1.(68) At the same time, some secretory proteins specific to inhibitory neurons were decreased, such as RELN, ERBB4, SST and NPY. (Additional file 1: Fig. S6) These suggest that upregulated changes involve multitude of functions, whereas downregulated genes implicated in E/I balance are the most notable signatures of Nes-KO PCN.
Linking Pten haploinsufficiency to intelligence, cognitive function, and schizophrenia
Next, we explored the potential neurological disorders and traits associated with Pten haploinsufficiency. First, the expression correlations between heterozygous and knockout PCN and AST were compared by Spearman correlation analysis (Fig. 6A-B). The results showed that the DEGs between Pten+/− and Nes-KO in PCN are significantly correlated (ρ = 6.1e-06, R = 0.68) (Fig. 6A), but not so in AST (Fig. 6B). Next, we curated genelists by integrating DEGs with DEPs from SSC of P30M Pten+/− mice. Comparing DEG of individual neural cells to DEP, Nes-KO AST has greatest number of overlapping, followed by Nes-KO PCN, (Fig. 6C-D; additional file 2: Table S3a & b). Overall, there is a lack of concordance between PCN DEGs and SSC DEPs. It could be due to the differences between neuronal cells in vitro and in vivo. Alternatively, PTEN complete depletion highly distorted the gene expression profiles as compared to Pten+/− conditions and could not be registered as DEPs in Pten+/− SSC.
Finally, to uncover the potential neurological disorders and traits associated with Pten haploinsufficiency, we curated gene lists by integrating DEPs from Pten+/− SSC and DEGs from Pten+/− and Nes-KO neural cells by filtering out non-DEP genes from DEGs of neural cells as NPC(DEP), AST(DEP), PCN(DEP) and translated DEGs which comprised of DEGs from all neural cells overlapped with DEPs (Additional file 2: Table S3c). Next, we performed enrichment analysis between the gene lists and genetic variants associated with neurological disorders and traits obtained from GWAS (Additional file 2: Table S3d).(69) Overall, we found significant enrichment (FDR < 0.05) of both DEP and translated DEGs on the traits of intelligence and cognitive function, and schizophrenia was significantly enriched with DEP (Fig. 6E; additional file 2: Table S3e). Further investigation at cell-type level, we observed AST (DEP) is significantly associated with intelligence and depression.