Alzheimer's disease is a neuropsychiatric and neurodegenerative disorder with growing incidence rate all over the world. This study signifies the identification of novel genes or proteins involved in pathophysiological aspects such as protein aggregation and neuroinflammation. The altered biological and molecular activities of DEGs as well as their signaling pathways are responsible for the development of AD. Furthermore, these genes can be targeted for therapeutic approaches in the treatment of AD. In this study we evaluated the DEGs from AD patients as compared to normal senile individuals that were extracted from the GEO database. A total of 1674 DEGs were identified in AD as well as in control blood samples, including both upregulated and downregulated genes and were used for the construction and analysis of the protein-protein interaction network. Notably, this extended network was built using the STRING database and has 744 nodes and 1992 edges. Furthermore, a giant or core network with 676 nodes and 1995 edges was created from the extended network. In addition, there are many subnetworks with fewer nodes and edges in the extended network. The first subnetwork has 5 nodes and 4 edges, whereas the second subnetwork has 4 nodes and 3 edges, and the third network has 3 nodes and 2 edges. The genes present in the subnetwork do not appear to be substantially related to disease severity. However, core network analysis is the main route for identifying truncated proteins by chaperones in AD pathogenesis. According to functional enrichment studies, genes in the core network are enriched in many biological processes such as GO:0006915; apoptotic process, GO:0045893; positive regulation of transcription, DNA-templated, GO:0006468; protein phosphorylation, GO:0046777; protein autophosphorylation, GO:0002181; cytoplasmic translation, GO:0007165; signal transduction, GO:0035556; intracellular signal transduction, GO:0006954; inflammatory response, GO:0042981; regulation of apoptotic process and GO:0097191; extrinsic apoptotic signaling pathway. These enriched biological functions support prior studies suggesting oligomeric amyloid beta buildup causes widespread protein phosphorylation, which leads to prolonged autophosphorylation of membrane receptor kinases. The activation of the GSK-3 beta signaling pathway as a result of autophosphorylation, destabilizes the microtubules, leading to the production of neurofibrillary tangles as downstream pathogenesis of AD. The KEGG pathway also indicated that all the DGEs in the core network are majorly involved in the downstream signaling of cancer, multiple neurodegenerative disorders, lipid and atherosclerosis, Salmonella infection, Chemokine signaling pathway, Human immunodeficiency virus 1 infection, Coronavirus disease-COVID-19, Human papillomavirus infection, and tuberculosis. These signaling pathways or disease conditions enhance the pro-inflammatory signaling pathways, resulting in severe synaptic dysfunction and/or neuronal death. The DEGs enriched in multiple neurodegenerative disorders, including AD are shown in Fig. 7.
In order to examine highly interacting pathways related to the pathophysiology of AD, the key nodes in the core network that are significantly interacting were identified. In accordance with the cutoff value determined in relation to the sum of the mean and standard deviation, we obtained 44 genes from the high BC value (cut off value = 0.019528) and 81 genes from the high degree value (cut off value = 12.66321). Further, we investigated the common genes present in both high BC and high degree; we found 32 genes, with the top six genes being HSP90AA1, RPS27A, CREBBP, UBC, GRB2, and MAPK3. It is further confirmed by presence of all these 6 genes in the hub genes (i.e., top 10 genes) in the core network were identified based on the five ranking methods of Cytohubba. Additionally, using the 5% of high BC genes in the core network, a backbone network was built to further validate the information flow in the core network. A total of 34 genes (5%) were selected from 676 genes from the core network and were pluged into the funrich software for functional enrichment analysis. In the backbone network majority of the genes are involved in the biological process such as signal transduction (41.2%), cell communication (29.4%), protein metabolism (23.5%) and followed by regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism (23.5%). Further, these backbone network genes were again filtered to the Cytoscape for the topological analysis, which returned HSP90AA1in the first position in all three parameters such as high degree, high BC and from cytohubba. Additionally, we got HSPA5 in the second position with high BC and cytohubba. Further, we compelled the presence of all the top genes from core network and backbone network analysis. We obtained HSP90AA1, UBC-RPS27A (closely associated genes), CREBBP, GRB2, and MAPK3, and TRAF6 as the major biomarkers enriched in AD pathogenesis. However, HSPA5 is a known biomarker in AD. Both HSP90AA1 and HSPA5, it is mainly dependent on ATP in the regulation and clearance of protein aggregation. However, it is not effective against fibril conformations as seen in AD and this prompts to increase in secretion and accumulation, which further substantiates the degeneration of neurons and glial cells.
As a newly identified HSP90AA1 biomarker, it is quite prevalent in eukaryotic cells, accounting for 1–2% of total cellular proteins. This protein's concentration can rise to as high as 4–6% in stressful situations. Funrich functional enrichment analysis also shows that the biological process of HSP90AA1 is involved in protein metabolism (GO:0019538) and its protein domain signifies the presence of coiled-coil region and HATPase c. Also, which is having fold chain enrichment of 101.92 in vRNP Assembly, 22.73 in eNOS activation, 21.04 in VEGFR1 specific signalling, 15.23 in glucocorticoid receptor regulatory network, 13.64 in Sema3A PAK dependent axon repulsion and 12.36 in IL-2 signaling events mediated by PI3K. In this regard, aberrantly expressed molecular chaperones have critical functions in regulating the aggregation of damaged proteins in cells, such as amyloid beta 1−42. This critically misfolded or aggregated protein induces tau phosphorylation as downstream pathogenesis. Some studies suggested that inhibition of over-expressed HSP90AA1 reduces the kinases activity and limits the tau phosphorylation as seen in AD (Ou et al., 2014). Furthermore, HSP90-induced amyloid beta 1−42 also activates the glial cells such as astrocytes and microglia and exuberates the inflammatory response leading to severe neuronal degeneration. Besides, HSP90 inhibitory studies show a promising role in the downregulation of several inflammatory responses, mainly by promoting the ubiquitin-protease pathway (Dukay et al., 2019; He et al., 2019) and as such, targeting HSP90AA1 in AD may reduce the pathophysiology by lowering alerted protein structure accumulation
RPS27A - Ubiquitin-40S ribosomal protein, which is also found to be intricated in protein metabolism, has UBQ protein domain (Montellese et al., 2020). It is involved in the biological process such as IRAK2 mediated TAK1 activation with 60.99-fold change, Fanconi anemia pathway with 50.89-fold change, NF-kB activation with 46.92-fold change, p75NTR signalling complexes with 46.92-fold-change, NRIF signaling with 43.57-fold change, and TRAF6 mediated induction of TAK1 complex with 43.57-fold change. Since, the presence of ubiquitin immunoreactivity in AD-related neuronal aggregates, the ubiquitin-proteasome system (UPS) was thought to have a role in the disease's progression (Gong et al., 2016). According to a recent study, a mutant version of UBC called Ub + 1 is connected to both the early and late stages of AD, which are characterized by synaptic dysfunction and neurodegeneration. When non-DNA-encoded dinucleotide deletion (s) are located in mRNA within or close to GAGAG motifs, this process is known as molecular misreading and results in the expression of Ub + 1 (Lam et al., 2000). In addition, it is also possible that other ubiquitin-proteosome members' dysfunction may also promote Aβ accumulation. Altogether, oxidative stress and Aβ accumulation induce the mutant ubiquitin, which was shown to reduce the activity of the proteasome in vitro. However, although UBB + 1 also increased the expression of heat shock proteins (Ding & Keller, 2001).
It has long been understood that cyclic-AMP response element binding protein (CREB), a protein that binds to the cyclic-AMP response element, is crucial for the conversion of short-term memory to long-term memory, which is mediated by long-lasting alterations in synaptic plasticity (Alberini, 2009). It is involved in biological processes, specifically the regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism, and it has protein domain structures such as a coiled-coil region, a zinc finger region, a bromodomain, and KAT1. These protein domains are majorly involved in protein acetylation, which can significantly modify a protein's surface characteristics, solubility, hydrophobicity, and ability to function. Also, it is involved in biological processes such as TRAF3-dependent IRF activation pathway with 20.46-fold change, NICD traffics to the nucleus with 18.60-fold change, notch-HLH transcription pathway with 18.60-fold change, retinoic acid receptors-mediated signaling with 17.30-fold change, and TRAF6 mediated IRF7 activation with 16.30-fold change. Recently, CREB signaling has been linked to several pathological states of the brain, such as cognitive and neurodegenerative illnesses, including AD. During normal conditions, protein kinase A phosphorylates CREBBP in the nucleus and this helps in the production of BDNF (Miranda et al., 2019). In AD, amyloid beta induces the extensive phosphorylation of tyrosine receptors, which results in the successive activation of phosphatases 1 and 2, which attenuates the activation site of CREBBP (serine 133 residue) and results in a lowered level of BNDF, neuronal excitability and plasticity, and triggers neurodegeneration (Wang et al., 2018). As a result, CREBBP levels can be used as a biomarker for unconsolidated long-term memory potentiation in Alzheimer's patients.
An adaptor protein, growth factor receptor-bound protein 2 (Grb2), in accordance with the Funrich functional enrichment analysis these proteins take part in biological processes such as Signal transduction, regulation of cell cycle, and Apoptosis in AD. Its role in biological pathway includes Spry regulation of FGF signaling with 38.18-fold change, signal attenuation with 37.03-fold change, signal regulatory protein (SIRP) family interactions with 37.03-fold change, negative regulation of FGFR signaling with 35.89-fold change and SHC-mediated cascade with 34.08-fold change. Also, it has SRC homology 2 & 3 as the major protein domains. As correlating with the enriched biological pathways, the phosphotyrosine-binding domain (PTB) and src homology domain (SH2) of the adaptor proteins Shc and Grb2 are able to directly bind tyrosine-phosphorylated APP. This is followed by the MAP kinase cascade's SoS, ras, Raf, and MEK are recruited, which activates ERK1/2 (Nizzari et al., 2012). Direct binding to APP or recruitment by Shc are two possible ways that Grb2 can take part in this process. This method of inducing ERK1/2 activity change may help to explain why AD patients' neurons begin to degenerate. Besides, the pathogenic correlation between Shc/Grb2 binding to Aβ during AD development is supported by the observation that the complexes Aβ/ShcA or Grb2 are significantly increased in AD brain compared to controls (Meister et al., 2013).
TRAF6 is increasingly being linked to conditions affecting the central nervous system, including neurodegenerative disorders and neuropathic pain. It was discovered to be integrated with various kinases to control signaling pathways and function as an E3 ubiquitin ligase. Also, it is found to be involved in many biological pathways, including IRAK2 mediated activation of TAK1 complex with 60.99-fold change, NF-kB and signal survival with 46.92-fold change, p75NTR recruits signaling complexes with 46.92-fold change, and induction of TAK1 complex signaling with 43.57-fold change. Its enriched protein domains include coiled coil region, ring structure, and Math structure. In AD, TRAF6 activates Becline1 (autophagy activator protein) through TLR4, resulting in ubiquitination of Beclin-1. This prevents autophagy and induces an inflammatory response. Furthermore, numerous activated astrocytes and microglia near amyloid plaques in Alzheimer's disease lead to the generation of inflammatory cytokines including IL-1B and TNF-α, and TRAF6 is critical to this process (Dou et al., 2018).
Considering the significant role of identified biomarkers in the pathological process of AD, eXpression2Kinases serves the objective of identification of transcriptional factors and their kinases., We discovered that,
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the transcriptional regulator TAF1 for HSP90AA1, HSPA5, and RPS27A is phosphorylated by CK2ALPHA, CDK1, GSK3B, and CDK2 kinases.
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HIPK2, CSNK2A1, CDK1, AKT1, GSK3B, PRKACA, RPS6KA3, RPS6KA1, and PKBALPHA kinases control the HSPA5 transcription regulator CREB1.
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CK2ALPHA, MAPK1, MAPK3, MAPK14, CDK1, AKT1, and GSK3B kinases control the HSPA5 transcription regulator SP1.
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RUNX1, a transcriptional regulator of GRB2 and CREBBP, is controlled by HIPK2, MAPK1, MAPK3, CSNK2A1, MAPK14, CDK1, GSK3B, and MAPK8 kinases.
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CDK1, AKT1, and GSK3B kinases regulate transcription regulator GATA2 for GRB2 and CREBBP.
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CREBBP transcription regulator RCOR1 is regulated by CK2alpha and CDK2 kinases.
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RPS27A transcription regulator SPI1 is regulated by CK2ALPHA, CSNK2A1, CK2, ABL1 and PRKCA kinases.