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Background: Interactions between the gut microbiota, microglia, and aging may modulate Alzheimer's disease (AD) pathogenesis but the precise nature of such interactions is not known., Methods: We developed an integrated multi-dimensional, knowledge-driven, systems approach to identify interactions among microbial metabolites, microglia, and AD. Publicly available datasets were repurposed to create a multi-dimensional knowledge-driven pipeline consisting of an integrated network of microbial metabolite-gene-pathway-phenotype (MGPPN) consisting of 34,509 nodes (216 microbial metabolites, 22,982 genes, 1329 pathways, 9982 mouse phenotypes) and 1,032,942 edges., Results: We evaluated the network-based ranking algorithm by showing that abnormal microglia function and physiology are significantly associated with AD pathology at both genetic and phenotypic levels: AD risk genes were ranked at the top 6.4% among 22,982 genes, P < 0.001. AD phenotypes were ranked at the top 11.5% among 9982 phenotypes, P < 0.001. A total of 8094 microglia-microbial metabolite-gene-pathway-phenotype-AD interactions were identified for top-ranked AD-associated microbial metabolites. Short-chain fatty acids (SCFAs) were ranked at the top among prioritized AD-associated microbial metabolites. Through data-driven analyses, we provided evidence that SCFAs are involved in microglia-mediated gut-microbiota-brain interactions in AD at both genetic, functional, and phenotypic levels., Conclusion: Our analysis produces a novel framework to offer insights into the mechanistic links between gut microbial metabolites, microglia, and AD, with the overall goal to facilitate disease mechanism understanding, therapeutic target identification, and designing confirmatory experimental studies., (© 2021. The Author(s).)

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles (NFTs), senile plaques (SPs), and a progressive loss of neuronal cells in selective brain regions. Rab10, a small Rab GTPase involved in vesicular trafficking, has recently been identified as a novel protein associated with AD. Interestingly, Rab10 is a key substrate of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine protein kinase genetically associated with the second most common neurodegenerative disease Parkinson's disease. However, the phosphorylation state of Rab10 has not yet been investigated in AD. Here, using a specific antibody recognizing LRRK2-mediated Rab10 phosphorylation at the amino acid residue threonine 73 (pRab10-T73), we performed immunocytochemical analysis of pRab10-T73 in hippocampal tissues of patients with AD. pRab10-T73 was prominent in NFTs in neurons within the hippocampus in all cases of AD examined, whereas immunoreactivity was very faint in control cases. Other characteristic AD pathological structures including granulovacuolar degeneration, dystrophic neurites and neuropil threads also contained pRab10-T73. The pRab10-T73 immunoreactivity was diminished greatly following dephosphorylation with alkaline phosphatase. pRab10-T73 was further found to be highly co-localized with hyperphosphorylated tau (pTau) in AD, and demonstrated similar pathological patterns as pTau in Down syndrome and progressive supranuclear palsy. Although pRab10-T73 immunoreactivity could be noted in dystrophic neurites surrounding SPs, SPs were largely negative for pRab10-T73. These findings indicate that Rab10 phosphorylation could be responsible for aberrations in the vesicle trafficking observed in AD leading to neurodegeneration.

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that are known to contribute to the adaptation of the heptahelical G protein-coupled receptors (GPCRs) and to regulate downstream signals through these receptors. GPCRs mediate the action of messengers that are key modulators of cardiac and vascular cell function, such as growth and differentiation. GRKs are members of a multigene family, which are classified into three subfamilies and are found in cardiac, vascular and cerebral tissues. Increasing evidence strongly supports the hypothesis that vascular damage is an early contributor to the development of Alzheimer disease (AD) and/or other pathology that can mimic human AD. Based on this hypothesis, and since kinases of this family are known to regulate numerous receptor functions both in the brain, myocardium and elsewhere, we explored cellular and subcellular localization by immunoreactivity of G protein-coupled receptor kinase 2 (GRK2), also known as beta-adrenergic receptor kinase-1(betaARK1), in the early pathogenesis of AD and in ischemia reperfusion injury models of brain hypoperfusion. In the present study, we used the two-vessel carotid artery occlusion model, namely the 2-VO system that results in chronic brain hypoperfusion (CBH) and mimics mild cognitive impairment (MCI) and vascular changes in AD pathology. Our findings demonstrate the early overexpression of GRK2 member kinase in the cerebrovasculature, especially endothelial cells (EC) following CBH, as well as in select cells from human AD tissue. We found a significant increase in GRK2 immunoreactivity in the EC of AD patients and after CBH, which preceded any amyloid deposition. Since GRK2 activity is associated with certain compensatory changes in brain cellular compartments and in ischemic cardiac tissue, our findings suggest that chronic hypoperfusion initiates oxidative stress in these conditions and appears to be the main initiating injury stimulus for disruption of brain and cerebrovascular homeostasis and metabolism.

The pathogenesis, which is primarily responsible for Alzheimer's disease (AD) and cerebrovascular accidents (CVA), seems to involve chronic hypoperfusion. The role of hypoperfusion, as a key factor for vascular lesions that causes oxidative stress, appears to be widely accepted as an initiator of AD. Specifically, accumulated oxidative stress increases vascular endothelial permeability and promotes leukocyte adhesions, which is coupled with alterations in endothelial signal transduction and redox-regulated transcription factors. Based on these recent findings, we hypothesize that the cellular and molecular mechanisms by which hypoperfusion-induced reactive oxygen species (ROS) accumulation impairs endothelial barrier function and promotes leukocyte adhesion induces alterations in normal vascular function and results in the development of AD. We are theorizing that mitochondria play a key role in the generation of ROS, resulting in oxidative damage to neuronal cell bodies, as well as other cellular compartment in the AD brain. All of these changes have been found to accompany AD pathology. We have studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD, yeast artificial chromosome (YAC) and C57B6/SJL transgenic positive (Tg+) mice overexpressing amyloid beta precursor protein (AbetaPP). In situ hybridization using mitochondrial DNA (mtDNA) probes for human wild and 5 kb deleted types and mouse types was performed along with immunocytochemistry using antibodies against amyloid precursor protein (APP), 8-hydroxy-2'-guanosine (8-OHG) and cytochrome c oxidase (COX). There was a higher degree of amyloid deposition, overexpression of oxidative stress markers, mitochondria DNA deletion and mitochondrial structural abnormality in the vascular walls of the human AD, YAC and C57B6/SJL Tg (+) mice compared to age-matched controls. Therefore, selective pharmacological intervention, directed for abolishing the chronic hypoperfusion state, would possibly change the natural course of development of dementing neurodegeneration.

Nitric oxide (NO) is a short-life key bioregulatory active molecule in the cardiovascular, immune and nervous systems. NO is synthesized by converting L-arginine to L-citrulline by enzymes called NO synthase (NOS). The growing body of evidence strongly supports the theory that this molecule appears to be one of the key targets for the disruption of normal brain homeostasis, which causes the development of brain lesions and pathology such as in Alzheimer's disease (AD) or other related dementia. The vascular content of NO activity appears especially to be a main contributor to this pathology before the over-expression of other NOS isoforms activity in a different brain cellular compartment. We speculate that pharmacological intervention using NO donors and/or NO suppressors will be able to delay or minimize the development of brain pathology and further progression of mental retardation.

We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.