Nedd4-1 regulates human sodium-dependent vitamin C transporter-2 functional expression in neuronal and epithelial cells.

The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is currently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we determined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways.

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-ß 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-ß 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.

Angiotensin receptor blockade with olmesartan alleviates brain pathology in obese OLETF rats.

Metabolic syndrome (MetS) is a rapidly increasing health concern during midlife and is an emerging risk factor for the development of neurodegenerative diseases, such as Alzheimer's disease (AD). While angiotensin receptor blockers (ARB) are widely used for MetS-associated hypertension and kidney disease, its therapeutic potential in the brain during MetS are not well-described. Here, we tested whether treatment with ARB could alleviate the brain pathology and inflammation associated with MetS using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. Here, we report that chronic ARB treatment with olmesartan (10 mg/kg/day by oral gavage for 6 weeks) partially but significantly ameliorated accumulation of oxidized and ubiquitinated proteins, astrogliosis and transformation to neurotoxic astrocytes in the brain of old OLETF rats, which otherwise exhibit the progression of these pathological hallmarks associated with MetS. Additionally, olmesartan treatment restored claudin-5 and ZO-1, markers of the structural integrity of the blood-brain barrier as well as synaptic protein PSD-95, which were otherwise decreased in old OLETF rats, particularly in the hippocampus, a critical region in cognition, memory and AD. These data demonstrate that the progression of MetS in OLETF rats is associated with deterioration of various aspects of neuronal integrity that may manifest neurodegenerative conditions and that overactivation of angiotensin receptor directly or indirectly contributes to these detriments. Thus, olmesartan treatment may slow or delay the onset of degenerative process in the brain and subsequent neurological disorders associated with MetS.

Valproic acid upregulates sodium-dependent vitamin C transporter-2 functional expression in neuronal cells.

Neuronal uptake of ascorbic acid (AA) in humans occurs via the human sodium-dependent vitamin C transporter-2 (hSVCT2). Recent studies show that a significantly lower level of vitamin C is present in the blood of epileptic patients. Consequently, focused studies investigating the involved molecular mechanisms for hSVCT2 regulation are vital to enhance vitamin C body homeostasis. Currently, little is known about the role of valproic acid (VPA), a drug utilized to treat epilepsy and a class I histone deacetylase inhibitor (HDACi), on AA uptake in neuronal systems. Thus, this study aims to examine the effect of VPA on hSVCT2 functional expression in neuronal cells. VPA treatment upregulated the AA uptake and this increased AA uptake was associated with a significant increase in hSVCT2 expression and SLC23A2 promoter activity in SH-SY5Y cells. Knockdown of HDAC2, a predominant isoform in neuronal systems, significantly increased hSVCT2 functional expression. VPA treatment in mice displayed increased mouse (m)SVCT2 protein, mRNA and heterogenous nuclear RNA (hnRNA) expression in the brain. In addition, Yin Yang-1 (YY1), a transcription factor that drives the SLC23A2 promoter activity, protein and mRNA expression levels were markedly upregulated in VPA-treated SH-SY5Y cells and mice brain. Together, our findings suggest that VPA upregulates the functional expression of SVCT2 via HDAC2 and transcriptional mechanism(s).

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.

Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake.

Vitamin C (ascorbic acid: AA) uptake in neurons occurs via the sodium-dependent vitamin C transporter-2 (SVCT2), which is highly expressed in the central nervous system (CNS). During chronic neuroinflammation or infection, CNS levels of lipopolysaccharide (LPS) and LPS-induced tumor necrosis factor-α (TNFα) are increased. Elevated levels of LPS and TNFα have been associated with neurodegenerative diseases together with reduced levels of AA. However, little is known about the impacts of LPS and TNFα on neuronal AA uptake. The objective of this study was to examine the effect of LPS and TNFα on SVCT2 expression and function using in vitro and in vivo approaches. Treatment of SH-SY5Y cells with either LPS or TNFα inhibited AA uptake. This reduced uptake was associated with a significant decrease in SVCT2 protein and mRNA levels. In vivo exposure to LPS or TNFα also decreased SVCT2 protein and mRNA levels in mouse brains. Both LPS and TNFα decreased SLC23A2 promoter activity. Further, the inhibitory effect of LPS on a minimal SLC23A2 promoter was attenuated when either the binding site for the transcription factor Sp1 was mutated or cells were treated with the NF-κB inhibitor, celastrol. We conclude that inflammatory signals suppress AA uptake by impairing SLC23A2 transcription through opposing regulation of Sp1 and NF-κB factors.

Assessing the activation/inhibition of tyrosine kinase-related pathways with a newly developed platform.

The phosphorylation of cellular proteins plays a crucial role in the transduction of various signals from outside the cell into the nucleus. The signals are transduced by phosphorylation chain reactions within multiple pathways; however, determining which pathways are responsible for each defined signal has proven challenging. To estimate the activity of each pathway, we developed a phosphorylation array platform comprising a protein array with 1200 proteins belonging to 376 signalling pathways and an analytical method to estimate pathway activity based on the phosphorylation levels of proteins. The performance of our system was assessed by reconstructing kinase-substrate relationships, as well as by estimating pathway activity upon epidermal growth factor (EGF) stimulation and the pharmacological inhibition of epidermal growth factor receptor (EGFR). As a result, kinase-substrate relationships were reliably reconstructed based on the precise measurement of phosphorylation levels of constituent proteins on the array. Furthermore, the pathway activities associated with EGF stimulation and EGFR inhibition were successfully traced through the related pathways from the outer membrane to the nucleus along a time course. Thus, our phosphorylation array system can effectively assess the activity of specific signalling pathways that are perturbed by extracellular stimuli, such as various drugs.

Generation of a humanized Aß expressing mouse demonstrating aspects of Alzheimer's disease-like pathology.

The majority of Alzheimer's disease (AD) cases are late-onset and occur sporadically, however most mouse models of the disease harbor pathogenic mutations, rendering them better representations of familial autosomal-dominant forms of the disease. Here, we generated knock-in mice that express wildtype human Aß under control of the mouse App locus. Remarkably, changing 3 amino acids in the mouse Aß sequence to its wild-type human counterpart leads to age-dependent impairments in cognition and synaptic plasticity, brain volumetric changes, inflammatory alterations, the appearance of Periodic Acid-Schiff (PAS) granules and changes in gene expression. In addition, when exon 14 encoding the Aß sequence was flanked by loxP sites we show that Cre-mediated excision of exon 14 ablates hAß expression, rescues cognition and reduces the formation of PAS granules.

Inflammatory Cytokine IL-1ß Downregulates Endothelial LRP1 via MicroRNA-mediated Gene Silencing.

Effective clearance of neurotoxic amyloid-beta (Aß) from the brain is a critical process to prevent Alzheimer's disease (AD). One major clearance mechanism is Aß transcytosis mediated by low-density lipoprotein receptor-related protein 1 (LRP1) in capillary endothelial cells. A marked loss of endothelial LRP1 is found in AD brains and is believed to significantly impair Aß clearance. Recently, we demonstrated that pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, significantly down-regulated LRP1 in human primary microvascular endothelial cells (MVECs). In this study, we sought to determine the underlying molecular mechanism by which IL-1ß led to LRP1 loss in MVECs. Reduced LRP1 protein and transcript were detected up to 24 h post-exposure and returned to the baseline levels after 48 h post-exposure with 1 ng/ml IL-1ß. This reduction was in part mediated by microRNA-205-5p, -200b-3p, and -200c-3p, as these microRNAs were concomitantly upregulated in MVECs exposed to IL-1ß. Synthetic microRNA-205-5p, -200b-3p, and -200c-3p mimics recapitulated LRP1 loss in MVECs without IL-1ß, and their synthetic antagomirs effectively reversed IL-1ß-mediated LRP1 loss. Importantly, we found that the expression of these three microRNAs was controlled by NF-κB as pharmacological NF-κB inhibitor, BMS-345541, inhibited the IL-1ß-mediated upregulation of these microRNAs and rescued LRP1 expression. siRNA-mediated silencing of IκB in MVECs elevated microRNA-200b-3p and decreased LRP1 transcript, partially confirming our overall findings. In conclusion, our study provides a mechanism by which pro-inflammatory IL-1ß instigates the suppression of LRP1 expression in MVECs. Our findings could implicate spatiotemporal loss of LRP1 and impairment of the LRP1-mediated clearance mechanism by endothelial cells.

Potential use of lenvatinib for patients with unresectable hepatocellular carcinoma including after treatment with sorafenib: Real-world evidence and in vitro assessment via protein phosphorylation array.

The efficacy and safety of lenvatinib (LEN) as a second/third-line treatment for unresectable hepatocellular carcinoma (HCC) after sorafenib (SOR) therapy remains unknown. We evaluated the outcomes of second/third-line LEN treatment, investigated the sensitivity of a SOR-resistant HCC cell line (PLC/PRF5-R2) to LEN, and assessed their signal transduction pathways by protein array analysis. We retrospectively enrolled 57 patients with unresectable HCC. Fifty-three radiologically evaluated patients comprised 34 molecular-targeted agent (MTA)-naive (first-line), nine intolerant to SOR (second-line), and 10 resistant to regorafenib (third-line). The objective response rates (ORRs) were 61.8% in first-line, 33.3% in second-line, and 20.0% in third-line groups. The overall survival (OS) in the first-line was significantly longer than that in the third-line group (p < 0.05). Patients with better liver functional reserves (child score, ALBI grade) exhibited higher ORR and longer OS. The IC50 of LEN against PLC/PRF5-R2 was significantly higher than that against PLC/PRF5. LEN significantly inhibited more LEN-related signal transduction pathways in PLC/PRF5 than in PLC/PRF5-R2 cells. This suggests that LEN is active and safe as a second/third-line treatment for unresectable HCC. LEN seems more effective for patients with HCC with better hepatic reserve functions or before MTA-resistance is acquired because of the partial cross-resistance to SOR.

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

BACKGROUND: Copper (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. METHODS: In 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. RESULTS: We 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. CONCLUSION: Our 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.

Genetic Ablation of Hematopoietic Cell Kinase Accelerates Alzheimer's Disease-Like Neuropathology in Tg2576 Mice.

Microglial dysregulation, pertaining to impairment in phagocytosis, clearance and containment of amyloid-ß (Aß), and activation of neuroinflammation, has been posited to contribute to the pathogenesis of Alzheimer's disease (AD). Detailed cellular mechanisms that are disrupted during the disease course to display such impairment in microglia, however, remain largely undetermined. We hypothesize that loss of hematopoietic cell kinase (HCK), a phagocytosis-regulating member of the Src family tyrosine kinases that mediate signals from triggering receptor expressed on myeloid cells 2 and other immunoreceptors, impairs microglial homeostasis and Aß clearance, leading to the accelerated buildup of Aß pathology and cognitive decline during the early stage of neuropathological development. To elucidate the pivotal role of HCK in AD, we generated a constitutive knockout of HCK in the Tg2576 mouse model of AD. We found that HCK deficiency accelerated cognitive decline along with elevated Aß level and plaque burden, attenuated microglial Aß phagocytosis, induced iNOS expression in microglial clusters, and reduced pre-synaptic protein at the hippocampal regions. Our findings substantiate that HCK plays a prominent role in regulating microglial neuroprotective functions and attenuating early AD neuropathology.

miR-181a negatively modulates synaptic plasticity in hippocampal cultures and its inhibition rescues memory deficits in a mouse model of Alzheimer's disease.

MicroRNAs play a pivotal role in rapid, dynamic, and spatiotemporal modulation of synaptic functions. Among them, recent emerging evidence highlights that microRNA-181a (miR-181a) is particularly abundant in hippocampal neurons and controls the expression of key plasticity-related proteins at synapses. We have previously demonstrated that miR-181a was upregulated in the hippocampus of a mouse model of Alzheimer's disease (AD) and correlated with reduced levels of plasticity-related proteins. Here, we further investigated the underlying mechanisms by which miR-181a negatively modulated synaptic plasticity and memory. In primary hippocampal cultures, we found that an activity-dependent upregulation of the microRNA-regulating protein, translin, correlated with reduction of miR-181a upon chemical long-term potentiation (cLTP), which induced upregulation of GluA2, a predicted target for miR-181a, and other plasticity-related proteins. Additionally, Aß treatment inhibited cLTP-dependent induction of translin and subsequent reduction of miR-181a, and cotreatment with miR-181a antagomir effectively reversed the effects elicited by Aß but did not rescue translin levels, suggesting that the activity-dependent upregulation of translin was upstream of miR-181a. In mice, a learning episode markedly decreased miR-181a in the hippocampus and raised the protein levels of GluA2. Lastly, we observed that inhibition of miR-181a alleviated memory deficits and increased GluA2 and GluA1 levels, without restoring translin, in the 3xTg-AD model. Taken together, our results indicate that miR-181a is a major negative regulator of the cellular events that underlie synaptic plasticity and memory through AMPA receptors, and importantly, Aß disrupts this process by suppressing translin and leads to synaptic dysfunction and memory impairments in AD.

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer.

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5-fluorouracil-based adjuvant therapy. In addition, the efficacy of 5-fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5-fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5-fluorouracil adjuvant chemotherapy.

CD4+ T-cell Immunity in the Peripheral Blood Correlates with Response to Anti-PD-1 Therapy.

Accumulating evidence indicates that CD8+ T cells in the tumor microenvironment and systemic CD4+ T-cell immunity play an important role in mediating durable antitumor responses. We longitudinally examined T-cell immunity in the peripheral blood of patients with non-small lung cancer and found that responders had significantly (P < 0.0001) higher percentages of effector, CD62Llow CD4+ T cells prior to PD-1 blockade. Conversely, the percentage of CD25+FOXP3+ CD4+ T cells was significantly (P = 0.034) higher in nonresponders. We developed a formula, which demonstrated 85.7% sensitivity and 100% specificity, based on the percentages of CD62Llow CD4+ T cells and CD25+FOXP3+ cells to predict nonresponders. Mass cytometry analysis revealed that the CD62Llow CD4+ T-cell subset expressed T-bet+, CD27-, FOXP3-, and CXCR3+, indicative of a Th1 subpopulation. CD62Llow CD4+ T cells significantly correlated with effector CD8+ T cells (P = 0.0091) and with PD-1 expression on effector CD8+ T cells (P = 0.0015). Gene expression analysis revealed that CCL19, CLEC-2A, IFNA, IL7, TGFBR3, CXCR3, and HDAC9 were preferentially expressed in CD62Llow CD4+ T cells derived from responders. Notably, long-term responders, who had >500-day progression-free survival, showed significantly higher numbers of CD62Llow CD4+ T cells prior to PD-1 blockade therapy. Decreased CD62Llow CD4+ T-cell percentages after therapy resulted in acquired resistance, with long-term survivors maintaining high CD62Llow CD4+ T-cell percentages. These results pave the way for new treatment strategies for patients by monitoring CD4+ T-cell immune statuses in their peripheral blood.

Intra- and extracellular ß-amyloid overexpression via adeno-associated virus-mediated gene transfer impairs memory and synaptic plasticity in the hippocampus.

Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as ß-amyloid (Aß) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aß either intra or extracellularly. We observed that expression of Aß in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aß inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aß in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aß oligomers both in vivo and in vitro.

Amyloid-beta impairs TOM1-mediated IL-1R1 signaling.

Defects in interleukin-1ß (IL-1ß)-mediated cellular responses contribute to Alzheimer's disease (AD). To decipher the mechanism associated with its pathogenesis, we investigated the molecular events associated with the termination of IL-1ß inflammatory responses by focusing on the role played by the target of Myb1 (TOM1), a negative regulator of the interleukin-1ß receptor-1 (IL-1R1). We first show that TOM1 steady-state levels are reduced in human AD hippocampi and in the brain of an AD mouse model versus respective controls. Experimentally reducing TOM1 affected microglia activity, substantially increased amyloid-beta levels, and impaired cognition, whereas enhancing its levels was therapeutic. These data show that reparation of the TOM1-signaling pathway represents a therapeutic target for brain inflammatory disorders such as AD. A better understanding of the age-related changes in the immune system will allow us to craft therapies to limit detrimental aspects of inflammation, with the broader purpose of sharply reducing the number of people afflicted by AD.

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 (Aß) 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 Aß and impairment of spatial memory, indicating that copper exposure has the pivotal role in the vascular damage and development of cognitive decline.

Metal Toxicity Links to Alzheimer's Disease and Neuroinflammation.

As the median age of the population increases, the number of individuals with Alzheimer's disease (AD) and the associated socio-economic burden are predicted to worsen. While aging and inherent genetic predisposition play major roles in the onset of AD, lifestyle, physical fitness, medical condition, and social environment have emerged as relevant disease modifiers. These environmental risk factors can play a key role in accelerating or decelerating disease onset and progression. Among known environmental risk factors, chronic exposure to various metals has become more common among the public as the aggressive pace of anthropogenic activities releases excess amount of metals into the environment. As a result, we are exposed not only to essential metals, such as iron, copper, zinc and manganese, but also to toxic metals including lead, aluminum, and cadmium, which perturb metal homeostasis at the cellular and organismal levels. Herein, we review how these metals affect brain physiology and immunity, as well as their roles in the accumulation of toxic AD proteinaceous species (i.e., ß-amyloid and tau). We also discuss studies that validate the disruption of immune-related pathways as an important mechanism of toxicity by which metals can contribute to AD. Our goal is to increase the awareness of metals as players in the onset and progression of AD.

Inhibition of hematopoietic cell kinase dysregulates microglial function and accelerates early stage Alzheimer's disease-like neuropathology.

Emerging evidence have posited that dysregulated microglia impair clearance and containment of amyloid-ß (Aß) species in the brain, resulting in aberrant buildup of Aß and onset of Alzheimer's disease (AD). Hematopoietic cell kinase (Hck) is one of the key regulators of phagocytosis among the Src family tyrosine kinases (SFKs) in myeloid cells, and its expression is found to be significantly altered in AD brains. However, the role of Hck signaling in AD pathogenesis is unknown. We employed pharmacological inhibition and genetic ablation of Hck in BV2 microglial cells and J20 mouse model of AD, respectively, to evaluate the impact of Hck deficiency on Aß-stimulated microglial phagocytosis, Aß clearance, and resultant AD-like neuropathology. Our in vitro data reveal that pharmacological inhibition of SFKs/Hck in BV2 cells and genetic ablation of their downstream kinase, spleen tyrosine kinase (Syk), in primary microglia significantly attenuate Aß oligomers-stimulated microglial phagocytosis. Whereas in Hck-deficient J20 mice, we observed exacerbated Aß plaque burden, reduced microglial coverage, containment, and phagocytosis of Aß plaques, and induced iNOS expression in plaque-associated microglial clusters. These multifactorial changes in microglial activities led to attenuated PSD95 levels in hippocampal DG and CA3 regions, but did not alter the postsynaptic dendritic spine morphology at the CA1 region nor cognitive function of the mice. Hck inhibition thus accelerates early stage AD-like neuropathology by dysregulating microglial function and inducing neuroinflammation. Our data implicate that Hck pathway plays a prominent role in regulating microglial neuroprotective function during the early stage of AD development.