During the current study, GSEA was performed to explore the expression profile of 19414 genes belonging to the BP of synapse assembly, synaptic vesicle cycle and neurotransmitter transport; as well as the KEGG pathways of phagosome, neuroactive ligand-receptor interaction and retrograde endocannabinoid signaling. It is worth noting that these genes in the processes and pathways were enriched in HD and early death individuals. Subsequently, a comprehensive characterization of regulatory network for the intersection genome of DEGs between HD and early death was integrated; hereby, the excavation of key target genes and interactive pathways from functional modules with respect to the trait of overall survival might contribute to elucidate the genome-scale pathogenesis of early death in HD patients.
Generally consistent with the results of GSEA, functional enrichment analysis exhibited DEGs of turquoise module participating in BP related to membrane potential regulation, nervous system process and chemical synaptic transmission; as well as KEGG pathways regarding GABAergic synapse, neuroactive ligand-receptor interaction and retrograde endocannabinoid signaling. Convincing previous evidence demonstrated that a series of huntingtin-interacting proteins associated with the deficits of GABAergic synapse yielded the onset of HD . Additionally, endocannabinoids modulated the synaptic transmission through the pathway of retrograde endocannabinoid signaling, resulting in cognitive impairment and movement dysfunction partially similar to the symptoms of HD . Based on MM > 0.9 and GS > 0.5 in WGCNA, seven DEGs including CA10, WSB2, NETO2, LPPR4, ACTR3B, PCDH19 and GABRB3 were identified as potential pathogenic factors for early death and HD. Among them, PCDH19 has been found to coordinate neurogenesis, neuronal migration and synaptic connections in mammalian cortex [27,28]. Low expression of PCDH19 promoted over-differentiation of progenitor cells, thus to premature neuronal apoptosis and structural defects of synapse; contrastively, high expression of PCDH19 improved abnormal neurogenesis, fine-scale spatial organization of excitatory neurons and synaptic junction in the cortex . ACTR3B, also known as actin related protein 3B, had implications in the nucleation and assembly of branched actin networks . It bound to DNA double-strand fractures and, in conjunction with actin, facilitating clustering and homologous directed repair at the breakage . There was evidence that the disturbance in ACTR3B expression was account for abnormal neuronal morphology and synaptic growth, as well as alterations in synapse activity [32-34], which was likely related to the pathogenesis of early death in HD.
Glaringly, in the global network analysis, GABRB3 was identified as a central hub gene at a site with genome-wide significant linkage to overall survival of HD. An observation from high-throughput flow cytometry assay revealed that GABRB3 was a critical gene for early development of central nervous system, and the functional impairment of GABRB3 was prone to neurodevelopment-related disorders . Specifically, it was increasingly expressed in the embryonic brain, with its encoded β3 subunit involved in the biological processes of stem cell differentiation, assembly and trafficking of GABA receptor [36,37]. The inhibitory synaptic transmission mediated by GABA receptor were susceptible to the accumulation of mutant huntingtin, leading to early degeneration of GABAergic neurons in the striatum of HD patients . Indeed, many of the manifestations that were emerged early in HD, such as hippocampal-dependent learning and memory impairment, were considerably attributed to the weakened inhibitory GABAergic transmission verified in a HD mouse model . This happened to coincide with the results of our network and GABAergic synapse pathway that low expression of the central hub gene GABRB3, interacting with other DEGs (e.g., GABRA1, GNB5 and KCNJ6), were included in the intersection genome between HD and early death, suggesting that GABRB3-mediated GABAergic synapse was probably a crucial pathway for early death of HD. With the exception of GABAergic synapse, the module-pathway network showed that GABRB3 also participated in neuroactive ligand-receptor interaction, morphine addiction and retrograde endocannabinoid signaling. The potential roles of these pathways in the overall survival of HD was not fully understood, and therefore deserved further exploration with an expectation of offering a GABRB3-related therapeutic target for this incurable disease.
Less is known about the properties of carbonic anhydrase CA10, rather than a non-catalytic activity of extracellular protein that has been described for two decades in relation to carbonic anhydrase . Nevertheless, recent study confirmed that CA10, an evolutionarily conserved ligand of neurexins in the surface transpose, served as a functional adaptor to enhance the expression of neurexin proteins, alter post-translational modifications and improve their surface levels . It mediated the interaction between neurexins and certain postsynaptic molecules, thereby facilitating the formation of novel trans-synaptic complexes . Evidence in zebrafish using morpholino-mediated knockdown of CA10 showed developmental retardation, aberrant behavior and early death under unknown mechanisms of CA10 deficiency . Herein, the network analysis exhibited multiple indirect interactions of CA10 with the center hub gene GABRB3, suggesting that GABRB3-mediated GABAergic transmission was possibly modulated by CA10 through its adaptor function. In episodic ataxia type-1families, carbonic anhydrase inhibitors decreasing the excitability of GABAergic interneurons by intracellular alkalinization were used to alleviate the clinical symptoms of cerebellar ataxia . Correspondingly, this would provide us with a feasible implication to the intervention of early death of HD by modulating CA10 to maintain postsynaptic potentials of GABAergic inhibition.
Visualization of the global regulatory network showed the down-regulation of candidate hub genes that were strongly related to the clinical trait of overall survival in HD patients. Further matrix of pairwise scatterplot confirmed a positively mutual correlation between them, implying that the expression of a gene subsided with the attenuation of the other. This might be illustrated by the involvement of candidate hub genes underlying the pathogenesis of early death in HD, particularly CA10, which indirectly interacted with GABRB3 in the pathway of GABAergic synaptic transmission [29,34,38,41]. Five genes (CA10, WSB2, ACTR3B, PCDH19 and GABRB3) with non-zero regression coefficients were selected to conduct the ROC analysis, of which the AUC values were all greater than 70%, supporting that they were hub genes and could be served as potential predictors for early death in HD worthy of clinical application . In addition, the verification of the Kaplan-Meier survival curve demonstrated that, compared with the high expression of CA10, WSB2, ACTR3B, PCDH19 and GABRB3, their low expression was associated with shorter overall survival of HD patients. It might be an intriguing hypothesis that the improvement of low expression of the hub genes was prospectively used for targeted treatment or prevention to HD patients with a high risk of early death. Since our prediction was based on an analysis of post-mortem samples, subsequent in vivo model experiments are required to validate the underlying mechanisms of the hub genes involved in the early death of HD.