In the existing studies, HAR1A has been confirmed to be related to the development and evolution of the human brain(16). The existed studies mainly explored the possible action pathway through bioinformatics analysis of human gene transcriptome(17). However, confirming the regulatory network of HAR1A by directly overexpressing HAR1A and observing the DEGs has not yet been reported. This study confirms that overexpressing HAR1A led to an obvious improvement in mice's cognition and memory. The possible pathways and mechanisms were explored through bioinformatics analysis.
Potential pathways of HAR1A affecting cortical development through CRs
HAR1A is specifically expressed in CRs, which existed in the developing human neocortex during gestational weeks 7–19 (18). CRs is crucial in the specification and migration of cortical neuron, suggesting that HAR1A plays an impotent role in neurogenesis(19). The results of GO analysis of DEGs showed that cerebral cortex development had the lowest p-value in all GO items related to brain development. Five differential genes were enriched in this biological process: Lhx2, Emx2, Foxg1, Nr2e1, Emx1.
FOXG1 encodes a transcription repression factor which is essential of the regional subdivision in the formation of the telencephalon and brain development(20). When FOXG1 is expressed in cortical progenitor cells, it stops the production of CRs by directly inhibiting a default transcriptional network(21). FOXG1-LHX2 interactions instruct the cessation of the CRs' production(22). CRs originate from the cortical hem21. Neurons in ventral pallium and cortical hem, which is in the medial pallium become subplate and CRs respectively after migrating and developing towards the neuroepithelium(23, 24). EMX1 and EMX2 are required to establish the Wnt-rich cortical hem domain(25, 26). EMX1 and EXM2 respectively encode a homeobox-containing transcription factor. They cooperate to promote the generation of CRs. NR2E1 participates in a feedback loop with the brain-specific microRNA, microRNA-9(miR-9), and Mouse miR-9 targets Foxg1 to control the generation of CRs in the medial pallium properly(27), (28), (29).
All DEGs enriched in Biological Process "cerebral cortex development" were involved in regulating CRs' function. CRs generated early in the cortex's marginal zone synthesize glycoprotein Reelin and secrete it into the peripheral intercellular stroma to regulate the morphology and biochemical maturation of radial glia cells (RGCs) (30), (31). RGCs' fibers act as scaffolds when newborn neurons migrate to their final destinations. Reelin is a stop signal of neurons migration, decides the neurons' orientation and positioning in their layers; abnormal function of CRs can lead to various neurological diseases, including Alzheimer's disease, Schizophrenia, Lissencephaly, Temporal lobe epilepsy(32).
HAR1A might affect CRs' function by regulating the expression of Lhx2, Emx2, Foxg1, Nr2e1, Emx1. HAR1A may affect cerebral cortex development in this way. The specific mechanism needs to be further studied.
HAR1A may affect synaptic function and formation and finally affects brain development and evolution.
The result of KEGG pathways showed that the neuroactive ligand-receptor interaction pathway showed the greatest significance, besides DEGs were notably enriched in neuroactive ligand-receptor interaction, axon guidance and cholinergic synapse. Results of GO analysis showed that DEGs were enriched in Glutamatergic synapse, synapse, memory, positive regulation of long-term synaptic potentiation. Multiple database analyses (GO, REAC, HPA) of DEGs showed the Synaptic signaling pathway and the presynapse pathway are very significant. Go analysis of 750 genes co-expressed with HAR1A in the datasets downloaded from TCGA showed that modulation of chemical synaptic transmission and regulation of trans-synaptic signaling were the top two significant enrichment items.
Based on the above analysis, it can be concluded that HAR1A take an important role in neuroactive ligand-receptor, synaptic and axon guidance. Those are all critical processes in the early development of the brain(33). Neurons migrate to proper locations to make connections by emitting axons during the development of the brain(34). Multiple environmental signals control these neurons to migrate and axons to grow. After axons get the appropriate position, synapses will be formatted for neurons' contact with each other(35). Axonal branches are over-formatted during the early development of the brain. Then specific neuronal connections are created by pruning the redundant synapses. (36),(37)Several ligand-receptor pairs are involved in each of these cellular events(38).
PPI analysis showed the co-expressed genes of HAR1A in humans made a regulation network, SNAP25, GRIN1, SYN1, DLG4, CAMK2A were the core genes of this network. SNAP25 produces a membrane protein on presynaptic plays an important role in neurotransmitter release. The protein encoded by SNAP25 is important in the docking and fusion of the vesicle membrane(39). The protein encoded by GRIN1 is an essential part of N-methyl-D-aspartate receptors, which belong to the glutamate receptor channel superfamily. They are heteromeric protein complexes with several subunits, which arrange into a certain spatial structure to form a ligand-gated ion channel. These subunits are crucial to the plasticity of synapses, which is the foundation of learning and memory(40). SYN1 belongs to the synapsin gene family, which encode neuronal phosphoproteins. It relates to the formation of the synaptic vesicles located on the cytoplasmic surface(41). DLG4 encodes a protein that belongs to the membrane-associated guanylate kinase (MAGUK) family. It is heteromultimeric with DLG2 (another MAGUK protein). DLG4 and DLG2 located on postsynaptic sites cooperate to form clustering receptors and ion channels by making a multimeric scaffold(42). CAMK2A encodes an alpha chain, which is essential for spatial learning and long-term hippocampal potentiation (LTP). It contains CaMK2A, NMDARs, and AMPARs which belong to the glutamatergic signaling family(43). They are essential for appropriate synaptic development and plasticity.
Briefly speaking, SNAP25 and SYN1 affect the transmission function of synapse; GRIN1, DLG4, CAMK2A encode proteins that makeup synapses; they involve in synapses' formation and plasticity. Existing evidence showed that HARs regulate neurodevelopmental processes that have diverged between humans and chimpanzees, such as synapse development(44). The human prefrontal cortex (PFC) is particularly enlarged more than other portions of the brain. It is related to the increasing of neocortical volume in primates. Changes in the synaptic distribution in the primate PFC may cause by different expression and transcription patterns of specific genes(45). Based on our experimental results and the conclusions of the existing studies, it can be speculated that HAR1A may affect the evolution of the human brain by regulating the expression of genes related to synaptic generation.
HAR1A regulates the crucial second signal calcium ion during development
Results of GO analysis of the DEGs showed that "Cellular response to calcium ion" of "biological process" exhibited the highest Rich factor value.
The calcium ion (Ca21) plays a crucial role in neurogenesis as an essential second messenger (46). Neural stem cells stay in the neocortex's ventricular zone to produce progenitor cells, glial cells and subsequently neurons, which build up the entire adult brain(47). Proliferation, migration, and differentiation are strictly regulated by multiple signals during establishing the accurate structures of the cerebral cortex, including cytosolic calcium ion (Ca21). The regulation of Ca2 + is essential in the delicate process of cortical development.