In this study, ten microarray gene expression datasets available in the public domain were analyzed using an integrated genomic approach. The datasets comprised 1446 brain samples from five brain regions, including the hippocampus, cerebellum, frontal, entorhinal and temporal cortices. Between the AD cases and the healthy controls, 1713 robust DEGs were identified, showing different dysregulated pathways in these brain regions. The down-regulation of the GABAergic synapse pathway and the retrograde endocannabinoid signaling pathway was common among the hippocampus, frontal, entorhinal and temporal cortices, all of which have been shown to be more impacted by AD, compared to the cerebellum.

A balance between neural excitation and inhibition is vital to proper brain function and its disruption contributes to several neuronal disorders such as AD. The down-regulation of the GABAergic synapse pathway and the decreased levels of Gamma-aminobutyric acid (GABA) in the hippocampus, frontal, entorhinal and temporal cortices found in this study have also been reported in other human and mice AD studies [35,36]. GABA is the main inhibitory neurotransmitter within the mammalian central nervous system [38]. A decreased level of GABA receptor subunits such as GABRA1, GABRA5, GABRB1, GABRB2, GABRB3, GABRG1, GABRG2 and GABRG3 have been observed at the transcriptional and protein levels in the hippocampus of AD patients [39,40,41,42,43].

However, in contrast to these results, the expression of another GABA receptor named GABRA6 was increased in cultured rat cerebellar granule neurons following treatment with amyloid beta (Aβ), one of the main pathological hallmark proteins implicated in AD [43,44]. Studies in both mice and humans have reported that the presence of Aβ can decrease the numbers and activity of GABA inhibitory interneurons, leading to impaired synaptic transmission and disrupted neural network activity, and resulting in cognitive impairment [45]. Interestingly, transplantation of GABA progenitor neurons into the hippocampus of mice overexpressing Aβ restored normal learning and memory [46].

Furthermore, the accumulation of phosphorylated tau (p-tau), another pathological hallmark protein of AD, in the hippocampus reduced extracellular GABA level and led to tau-induced anxiety in mice. In addition, p-tau accumulation in GABAergic interneurons in the dentate gyrus of AD patients and mice also caused disruption of GABAergic transmission [47,48]. This means that the GABAergic synapse pathway is down-regulated in AD, although there are some conflicting results from mice models [43]. However, AD has a complex neuropathological basis and mice models may not accurately represent underlying pathological events of AD in humans.

Another common down-regulated pathway between the AD sensitive brain regions was the retrograde endocannabinoid signaling pathway. The endocannabinoid system (ECS) is comprised of endocannabinoids (eCBs) that are endogenous lipid-based neurotransmitters, along with their receptors such as cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R), and the enzymes that are involved in their synthesis and degradation [49]. Arachidonoyl ethanolamide (anandamide or AEA) and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids [49]. ECS plays a vital role in the central nervous system function and in the regulation of the endocrine and immune systems [50,51]. Endocannabinoid-mediated retrograde signaling was first reported in 2001, where eCBs were shown to mediate a type of short-term synaptic plasticity known as depolarization-induced suppression of inhibition (DSI)/excitation (DSE) [52,53]. Later, the effect of eCBs on long-term depression (eCB-LTD) on both excitatory and inhibitory synapses was also observed [54]. Although the alteration of ECS components in AD animal models has remained controversial and mostly unchanged levels of these components in the hippocampus have been reported, beneficial effects of CB1R activation via exogenous cannabinoids has been observed against Aβ-induced neurotoxicity, particularly microglia activation in several cell models [54,55,56]. Moreover, eCBs have anti-inflammatory and neuroprotective properties, and since neuroinflammation is a key driver of neurodegenerative diseases such as AD, restoration of dysregulated ECS has emerged as a promising therapeutic option [55].

Interestingly, the down-regulation of retrograde endocannabinoid signaling also has been observed in a meta-analysis of gene expression data from patients with mild cognitive impairment (MCI) [57]. MCI patients are at a higher risk of developing AD as MCI is considered an early stage of AD. Thus, the molecular changes in the respective brain regions of MCI patients may represent early changes that mediate the development of AD [57,58]. The down-regulation of the retrograde endocannabinoid system in MCI patients, and in the four AD sensitive brain regions but not in the cerebellum of AD patients, highlighted dysregulation of this pathway as an early and key change in AD development. As such, this can be a promising therapeutic target for early intervention against AD progression.

The miRNAs and TFs that interact with robust DEGs in the GABAergic synapse pathway and in retrograde endocannabinoid signaling were also investigated here. Among the identified miRNAs, hsa-mir-17-5p, hsa-mir-106a-5p and hsa-mir-373-3p were found to interact with robust DEGs of both pathways. Recently, the elevated levels of hsa-mir-17-5p have been reported in the human AD brain and the microglia of AD mice models, coupled with the down-regulation of autophagy markers, that consequently impaired Aβ clearance [59]. In contrast to our results, decreased serum level of hsa-mir-106a-5p has previously been observed in AD patients [60].

However, recent studies have reported that hsa-mir-106a-5p decreased the level of VEGFA, which plays a protective role against cognitive impairment in AD patients, and its level is decreased in AD patients [61,62]. Therefore, the increased level of hsa-mir-106a-5p in AD patients may be involved in cognitive impairment, and thus its abundance may be altered in patients with mild cognitive impairment. No data is yet available in the published literature on the role of hsa-mir-373-3p in AD; however, a recent study indicated that hsa-mir-373-3p inhibits epithelial–mesenchymal transition in choriocarcinoma through targeting TGFβR2 [63]. Interestingly, the level of TGFβR2 in AD patients was shown to be about 50% lower than normal [64], therefore hsa-mir-373-3p may be associated with decreased level of TGFβR2 in AD patients. Collectively, there are limited data about these miRNAs and further studies are needed to establish their role in AD.

In order to investigate the upstream regulation of robust DEGs, TF analysis was performed. This returned the three TFs, ELK1, GATA1 and GATA2 that interact with robust DEGs involved in both the GABAergic synapse pathway and the retrograde endocannabinoid signaling pathway. ELK-1 belongs to the ternary complex factor (TCF) subfamily of ETS-domain TFs and its presence in diverse brain regions has been previously reported [65]. ELK-1 plays a dual role in neuronal function; while its expression is required for proper neuronal differentiation, overexpression and distinct phosphorylation of this TF is toxic for neuronal cells [66]. ELK-1 decreases expression of the presenilin 1 gene (PS1), which is involved in the proteolytic processing of the amyloid precursor protein (APP) and the generation of amyloidogenic Aβ, while, in turn, the presence of Aβ decreases ELK-1 activation and may derepress PS1 expression [66,67,68]. The other two identified TFs, GATA1 and GATA2, belong to the GATA family. Aberrant expression of GATA1 has been reported in AD patients previously, and has been shown to induce the expression of the β-amyloid precursor mRNA, and modulate γ-secretase activity. GATA1 may therefore be involved in the impairment of the synaptic plasticity and cognitive function [69,70]. GATA2, on the other hand, has been reported to play a key role in neuroglobin (NGB) activation, which has been shown to exhibit protective effects for neuronal cells in AD [71]. The expression of NGB is increased in early and moderate stages of AD but significantly decreased in advanced stages, which might be due to the decreased levels of GATA2 in the advanced stages of AD [72,73]. However, further studies are needed to evaluate this association. Interestingly, previous analysis of DEGs between the entorhinal cortex of AD patients and healthy controls also showed alteration of the retrograde endocannabinoid signaling pathway and predicted GATA1 and GATA2 as the upstream regulators of the identified DEGs in AD [74]. Our results here identified altered pathways in each brain region of AD patients and shared pathways between AD sensitive brain regions. Furthermore, our study also predicted miRNAs and TFs that interact with the DEGs involved in these shared pathways. However, experimental data about these regulators are limited and further studies are needed to reveal their role in AD pathogenesis.