Sex differences in cell type-specific transcriptional programs and GPCR signaling in the human subgenual anterior cingulate cortex

Technical Summary: Sex Differences in Cell Type-Specific Transcriptional Programs and GPCR Signaling in the Human sgACC

Problem Statement
Psychiatric disorders exhibit marked sex differences in disease onset, symptom presentation, and treatment response. While alterations in specific cell types within the subgenual anterior cingulate cortex (sgACC) have been linked to psychiatric disease, it remains unclear whether intrinsic, sex-specific transcriptional profiles exist in these populations under baseline (non-psychiatric) conditions. Understanding these baseline differences is critical for explaining sex-biased vulnerability to neural circuit dysfunction and treatment variability. This study addresses the gap in knowledge regarding how sex differences manifest across molecularly distinct neuronal populations in the human sgACC.

Methodology
The study employed a multi-modal approach combining human postmortem transcriptomics with rodent electrophysiology:

1. Human Transcriptomics:

   • Sample: RNA-sequencing data from laser-capture microdissected (LCM) neurons from the sgACC of 19 non-psychiatric control subjects (9 females, 10 males).
   • Cell Types: Five distinct populations were analyzed: excitatory pyramidal neurons from layers 2/3 (PyrL2/3) and 5/6 (PyrL5/6), and inhibitory interneurons expressing parvalbumin (PVALB), somatostatin (SST), or vasoactive intestinal peptide (VIP).
   • Analysis:
     • Differential Expression (DE): Linear models (limma) identified sex-associated differentially expressed genes (DEGs) with thresholds of $p < 0.05$ and $|\log_2\text{FC}| > 0.26$.
     • Pathway Enrichment: Gene Ontology (GO) analysis via Metascape.
     • Network Analysis: Weighted Gene Co-expression Network Analysis (WGCNA) identified modules of co-expressed genes. Module Differential Connectivity (MDC) was used to compare network strength between sexes. Hub genes were identified using the ARACNe method.
     • Overlap: Rank-rank hypergeometric overlap (RRHO) assessed concordance/discordance of DE patterns across cell types.

2. Rodent Electrophysiology:

   • Model: Adult male and female PV-Cre $\times$ Rosa26-tdTomato mice to fluorescently identify PVALB interneurons in the prelimbic prefrontal cortex (PL-PFC).
   • Technique: Whole-cell patch-clamp recordings in current-clamp mode.
   • Protocol: Input-output curves were generated via current injections. The muscarinic agonist oxotremorine-M (OXO) was applied to activate GPCRs. Experiments included the M1 antagonist VU0255035 and the M2 antagonist AF-DX 116 to isolate M2 receptor effects.

Key Contributions and Results

• Cell-Type-Specific Transcriptional Sex Differences:

  • Sex differences in gene expression were detected across all five cell types but varied significantly in magnitude and direction.
  • PVALB Interneurons: Showed the strongest female enrichment (355 DEGs), with pathways related to synaptic and channel activity.
  • SST Interneurons: Exhibited strong male enrichment (461 DEGs), mapping to epithelial cell and tight junction processes.
  • VIP and Pyramidal Cells: Also displayed distinct sex-biased profiles, though with less overall enrichment than PVALB or SST.
  • Limited Convergence: RRHO analysis revealed minimal concordance in sex-biased gene expression across different cell types, indicating that transcriptional sex differences are largely cell-type-specific rather than global cortical phenomena.

• Sex-Specific Network Connectivity:

  • WGCNA identified preserved co-expression modules with sex-biased connectivity.
  • SST Modules: The SST_4 module was more connected in females and enriched for DEGs. Hub genes included immune signaling factors (IL1R1, DOCK2) and extracellular matrix components (MMP2). Notably, while the module was more connected in females, specific hub genes like IL1R1 were male-enriched.
  • PVALB Modules: The PVALB_1 module showed higher connectivity in males. Hub genes involved GPCR signaling (GPR75, GPR22, GNB3) and mitochondrial/bioenergetic processes (MRPL28, TIMM50, IDH2).

• GPCR Signaling and Functional Validation:

  • Transcriptomic Finding: Among GPCRs, CHRM2 (encoding the M2 muscarinic acetylcholine receptor) showed the second strongest sex difference overall and was selectively enriched in female PVALB cells (logFC = 3.7).
  • Functional Validation: Electrophysiology in mouse PFC demonstrated that M2 receptor activation (via OXO) significantly increased the maximum firing rates of PVALB interneurons in female mice but had no effect in male mice.
  • Mechanism Confirmation: The sex-specific increase in firing in females was abolished by the M2 antagonist AF-DX 116, confirming the effect is mediated by M2 receptors.

Significance and Claims
The authors claim that their findings demonstrate intrinsic, cell type-specific sex differences in the human sgACC that correspond to functional differences in neuromodulator signaling. Specifically:

1. Baseline Differences: Sex differences in transcriptomic profiles, biological pathways, and network connectivity exist in non-psychiatric individuals, suggesting these are fundamental biological variations rather than disease artifacts.
2. Functional Relevance: The study bridges human transcriptomics and rodent physiology, showing that the female enrichment of CHRM2 in PVALB cells translates to a functional sex difference in M2 receptor signaling (increased excitability in females).
3. Therapeutic Implications: Given that GPCRs are major drug targets and psychiatric conditions show sex-biased treatment responses, these findings suggest that sex-specific GPCR signaling (particularly M2 in PVALB cells) may underlie differences in circuit function and treatment efficacy in disorders like major depressive disorder and schizophrenia.

The authors maintain a modest tone regarding their claims, noting that the limited sample size required the use of raw p-values rather than FDR-corrected values for some interpretations and that future studies with larger cohorts and genetic manipulations are needed to confirm these mechanisms definitively.