## 3 Results and Discussion
cAMP-dependent signaling takes place in neuronal metabolism, growth cone motility, and neuroprotection in the central nervous system [31]. Reactome pathway analysis indicated that _Rapgef4_ was significantly enriched in Rap1 and the integrin signaling R-HAS pathway, platelet aggregation, and glycogen-like peptide (Figure S3C, Table S11). Neuronal Rap1 regulates energy balance, glucose homeostasis, and leptin actions [32]. Moreover, the results of GO analysis indicated that _Pax6_, a gene which the encoded highly conserved transcription factor is essential in the formation of tissues and organs during embryonic development, was significantly enriched in BP, including regulation of core promoter binding, glandular epithelial cell development, and neuron fate commitment (Figure 3A). _Pax6_ plays a critical role for neural stem cell proliferation and neurogenesis in many regions of the central nervous system, including the cerebral cortex [33]. It also controls neuronal development and targets a large number of promoters in neural progenitor cells [34]. Furthermore, the results of the Reactome pathway analysis showed that _Cask_ was mainly enriched in dopamine neurotransmitter release cycle R-HAS and assembly and cell surface presentation of NMDA receptors R-HAS (Figure 3B). The encoded multidomain scaffolding protein is highly expressed in the mammalian nervous system and participates in brain development [35]. It was originally identified as a binding partner of neurexins, transmembrane proteins expressed in neurons, and neuroendocrine cells, while loss of its action affects synaptic function in cortical excitatory neurons [36]. Recent studies indicated that CASK is localized to the nucleus in both mouse neuronal cultures and brain tissues [37]. Figure 3: GO term and pathway enrichment analysis performed using Enrichr on DEGs. (**A**) The top 10 enriched biological process for _Pax6_. (**B**) The top 10 enriched Reactome pathway for _Cask_. (**C**) The top 10 enriched cellular component for _Ctsb_. (**D**) The top 10 enriched biological process for _ZFHX3_. Cathepsin B (_CTSB_), another high-scored gene in our analysis, plays a neuroprotective role in AD and elevated levels have been associated with targeting intervention against the disease. It is considered as a candidate protease for the generation of N-terminally truncated A\(\beta\) in astroglial cell cultures [38]. Enhanced hippocampal and cortical amyloid depositions were also observed in the cathepsin B-deficient mouse model of AD overexpressing human amyloid protein precursors (hAPP) [39]. According to our GO analysis, MF showed that _Ctsb_ was enriched in cysteine-type peptidase activity (Figure S6B, Table S20). New insights into the role of cysteine protease inhibitor, such as cysteine cathepsin and calpain 1, in neuroinflammation have been recently reported [40; 41]. For CC, _Ctsb_ was mainly enriched in endolysosome lumen, endolysosome, and endosome lumen (Figure 3C). Alterations in endolysosomal trafficking can induce neurodevelopmental progression [42]. In particular, the accumulation of protease-deficient LAMP1-positive organelles has been observed in axonal distensions near extracellular A\(\beta\) plaques in AD modes, indicating defects in lysosome maturation with AD pathogenesis [43]. Lastly, the GO analysis showed that _Zfnx3_ in BF was significantly enriched in the regulation of neuronal differentiation, adhesion, and brain development (Figure 3D). _ZfHx3_ (zinc finger homeobox 3) encodes a transcription factor with multiple homeodomains and zinc finger motifs which regulates myogenic and neuronal differentiation. The survival of neurons by inducing platelet-derived growth factor receptor \(\beta\) expression is promoted through the signaling pathway involving cAMP-responsive element-binding protein (CREB) and ZFHX3 [44]. The KEGG pathway map (Figure S8) shows significant expressed genes identified in the discrimination of the cell classes [45].