Characterizing the Effect of Chronic Copper Exposure on Neuropathology and Induction of Gut Microbiome Dysbiosis in an APP Knock-In Model of Alzheimer's Disease

Chronic exposure to copper is a putative environmental risk factor for AD. Although copper is an essential metal, environmental exposure to its inorganic cupric form (Cu2+) may exert toxicity. However, exact neurotoxic mechanisms of action of copper, and its contribution to AD neuropathology remain largely unelucidated. Copper is a natural antimicrobial agent, and here, we investigate its impact of copper exposure on the gut microbiome and integrity as well as effects on cognition and AD pathology. Recent studies implicate gut dysbiosis in the onset and progression of AD in both humans and animal models. We believe that chronic exposure to environmentally relevant dose of copper through drinking water will reduce richness and diversity of the gut microbiota in both WT and hAPPNL-G-F knock-in (APP-KI) mice. Thus, we hypothesize that this copper-induced dysbiosis may perturb host metabolism and inflammatory homeostasis to contribute to AD neuropathology. In this dissertation, male and female APP-KI and wildtype C57BL/6J mice were exposed to 1.3 ppm CuCl2 in drinking water ad-libitum for 3(pilot study) and 9(chronic study) months. The present studies use a host of behavioral, immunohistochemical, biochemical and microbial analyses to investigate the effect of copper on microbiota, amyloidosis, neuroinflammation and cognition in AD mice. In our pilot study, we discovered that while there were no significant changes in AD neuropathology or inflammatory status in WT or APP-KI mice, we observed that WT mice microbiota were resilient to the effects of copper administration. However, beginning 1 month after initiation of exposure, APP-KI mice developed a significantly different microbial composition according to the Bray Curtis -diversity. This was largely driven by a decrease in Firmicutes, and specifically in the genus Allobaculum and an increase in the phylum Bacteroidetes, specifically in the genus S24-7. These changes mirror some of the changes observed in studies in other AD mouse models and AD patients. In our chronic study, spanning from after weaning and until the mice were 10 months, old, we discovered no significant changes in cognition and a mild change in the cytokine profile of AD mice, with a reduction of IL-4 in plasma, following copper administration. With this longer treatment paradigm, we did observe increased in amyloidosis, both dense core and diffuse plaques, with copper treatment. Further, along with evidence that plaque-associated microglia are of a diseased phenotype, the amount of these microglia was significantly increased with copper treatment indicating that copper perturbs neuroinflammation. In the gut microbiota, we observe unexpected results. Copper directs the developmental trajectory of WT mice but with time the microbiota of AD mice is not changed much. This may indicate differing windows of susceptibility to copper exposure between the genotypes of mice and between pilot study and the chronic study paradigms

Copper-Induced Upregulation of MicroRNAs Directs the Suppression of Endothelial LRP1 in Alzheimer's Disease Model.

Chronic exposure to copper and its dyshomeostasis have been linked to accelerated cognitive decline and potentially increasing risk for Alzheimer's disease (AD). We and others have previously demonstrated that exposure to copper through drinking water significantly increased parenchymal amyloid-beta (Aβ) plaques and decreased endothelial low-density lipoprotein receptor-related protein 1 (LRP1) in mouse models of AD. In this study, we determined the underlying mechanisms that microRNA critically mediated the copper-induced loss of endothelial LRP1. In human primary microvascular endothelial cells (MVECs), microRNA-200b-3p, -200c-3p, and -205-5p were significantly elevated within the 24-h exposure to copper and returned to baseline after 48-h postexposure, which corresponded with the temporal change of LRP1 expression in these cells. Transient expression of synthetic microRNA-200b-3p, -200c-3p, or -205-5p on MVECs significantly decreased endothelial LRP1, and cotreatment of synthetic antagomirs effectively prevented the loss of LRP1 during copper exposure, collectively supporting the key regulatory role of these microRNAs in copper-induced loss of LRP1. In mice, a significant reduction of LRP1 in cortical vasculature was evident following 9 months exposure to 1.3 ppm copper in drinking water, although the levels of cortical microRNA-205-5p, -200b-3p, and -200c-3p were only marginally elevated. This, however, correlated with increased vascular accumulation of Aβ and impairment of spatial memory, indicating that copper exposure has the pivotal role in the vascular damage and development of cognitive decline

Calsyntenin-3 interacts with the sodium-dependent vitamin C transporter-2 to regulate vitamin C uptake

Ascorbic acid (AA) uptake in neurons occurs via a Na+-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library. This interaction was confirmed by co-immunoprecipitation, mammalian two-hybrid (M2H), and co-localization in human cell lines. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells led to a marked increase in AA uptake. Reciprocally, siRNA targeting hCLSTN3 inhibited AA uptake. In the J20 mouse model of Alzheimer's disease (AD), mouse (m)SVCT2 and mCLSTN3 expression levels in hippocampus were decreased. Similarly, expression levels of hSVCT2 and hCLSTN3 were markedly decreased in hippocampal samples from AD patients. These findings establish CLSTN3 as a novel hSVCT2 interactor in neuronal cells with potential pathophysiological significance

Chronic copper exposure directs microglia towards degenerative expression signatures in wild-type and J20 mouse model of Alzheimer’s disease

BackgroundCopper (Cu) is an essential metal mediating a variety of vital biological reactions with its redox property. Its dyshomeostasis has been associated with accelerated cognitive decline and neurodegenerative disorders, such as Alzheimer's disease (AD). However, underlying neurotoxic mechanisms elicited by dysregulated Cu remain largely elusive. We and others previously demonstrated that exposure to Cu in drinking water significantly exacerbated pathological hallmarks of AD and pro-inflammatory activation of microglia, coupled with impaired phagocytic capacity, in mouse models of AD.MethodsIn the present study, we extended our investigation to evaluate whether chronic Cu exposure to wild-type (WT) and J20 mouse model of AD perturbs homeostatic dynamics of microglia and contributes to accelerated transformation of microglia towards degenerative phenotypes that are closely associated with neurodegeneration. We further looked for evidence of alterations in the microglial morphology and spatial memory of the Cu-exposed mice to assess the extent of the Cu toxicity.ResultsWe find that chronic Cu exposure to pre-pathological J20 mice upregulates the translation of degenerative genes and represses homeostatic genes within microglia even in the absence amyloid-beta plaques. We also observe similar expression signatures in Cu-exposed WT mice, suggesting that excess Cu exposure alone could lead to perturbed microglial homeostatic phenotypes and contribute to accelerated cognitive decline.ConclusionOur findings highlight the risk of chronic Cu exposure on cognitive decline and altered microglia activation towards degenerative phenotypes. These changes may represent one of the key mechanisms linking Cu exposure or its dyshomeostasis to an increased risk for AD

Exposure to quasi-ultrafine particulate matter accelerates memory impairment and Alzheimer’s disease-like neuropathology in the AppNL-G-F knock-in mouse model

Exposure to traffic-related air pollution consisting of particulate matter (PM) is associated with cognitive decline leading to Alzheimer's disease (AD). In this study, we sought to examine the neurotoxic effects of exposure to ultrafine PM and how it exacerbates neuronal loss and AD-like neuropathology in wildtype (WT) mice and a knock-in mouse model of AD (AppNL-G-F/+-KI) when the exposure occurs at a prepathologic stage or at a later age with the presence of neuropathology. AppNL-G-F/+-KI and WT mice were exposed to concentrated ultrafine PM from local ambient air in Irvine, California, for 12 weeks, starting at 3 or 9 months of age. Particulate matter-exposed animals received concentrated ultrafine PM up to 8 times above the ambient levels, whereas control animals were exposed to purified air. Particulate matter exposure resulted in a marked impairment of memory tasks in prepathologic AppNL-G-F/+-KI mice without measurable changes in amyloid-β pathology, synaptic degeneration, and neuroinflammation. At aged, both WT and AppNL-G-F/+-KI mice exposed to PM showed a significant memory impairment along with neuronal loss. In AppNL-G-F/+-KI mice, we also detected an increased amyloid-β buildup and potentially harmful glial activation including ferritin-positive microglia and C3-positive astrocytes. Such glial activation could promote the cascade of degenerative consequences in the brain. Our results suggest that exposure to PM impairs cognitive function at both ages while exacerbation of AD-related pathology and neuronal loss may depend on the stage of pathology, aging, and/or state of glial activation. Further studies will be required to unveil the neurotoxic role of glial activation activated by PM exposure