Inflammatory cytokine, IL-1β, regulates glial glutamate transporter via microRNA-181a in vitro

Glutamate overload triggers synaptic and neuronal loss that potentially contributes to neurodegenerative diseases including Alzheimer's disease (AD). Glutamate clearance and regulation at synaptic clefts is primarily mediated by glial glutamate transporter 1 (GLT-1). We determined that inflammatory cytokines significantly upregulated GLT-1 through microRNA-181a-mediated post-transcriptional modifications. Unveiling the key underlying mechanisms modulating GLT-1 helps better understand its physiological and pathological interactions with cytokines. Primary murine astrocyte and neuron co-culture received 20 ng/mL IL-1β, TNF-α, or IL-6 for 48 h. Soluble proteins or total RNA were extracted after treatment for further analyses. Treatment with inflammatory cytokines, IL-1β and TNF-α, but not IL-6, significantly increased GLT-1 steady-state levels (p≤0.05) without affecting mRNA levels, suggesting the cytokine-induced GLT-1 was regulated through post-transcriptional modifications. Among the candidate microRNAs predicted to modulate GLT-1, only microRNA-181a was significantly decreased following the IL-1β treatment (p≤0.05). Co-treatment of microRNA-181a mimic in IL-1β-treated primary astrocytes and neurons effectively blocked the IL-1β-induced upregulation of GLT-1. Lastly, we attempted to determine the link between GLT-1 and microRNA-181a in human AD brains. A significant reduction of GLT-1 was found in AD hippocampus tissues, and the ratio of mature microRNA-181a over primary microRNA-181a had an increasing tendency in AD. MicroRNA-181a controls rapid modifications of GLT-1 levels in astrocytes. Cytokine-induced inhibition of microRNA-181a and subsequent upregulation of GLT-1 may have physiological implications in synaptic plasticity while aberrant maturation of microRNA-181a may be involved in pathological consequences in AD

The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration

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-β (Aβ), 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 Aβ clearance, leading to the accelerated buildup of Aβ 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 Aβ level and plaque burden, attenuated microglial Aβ 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

Ceftriaxone ameliorates tau pathology and cognitive decline via restoration of glial glutamate transporter in a mouse model of Alzheimer's disease

Glial glutamate transporter, GLT-1, is the major Na+-driven glutamate transporter to control glutamate levels in synapses and prevent glutamate-induced excitotoxicity implicated in neurodegenerative disorders including Alzheimer's disease (AD). Significant functional loss of GLT-1 has been reported to correlate well with synaptic degeneration and severity of cognitive impairment among AD patients, yet the underlying molecular mechanism and its pathological consequence in AD are not well understood. Here, we find the temporal decrease in GLT-1 levels in the hippocampus of the 3xTg-AD mouse model and that the pharmacological upregulation of GLT-1 significantly ameliorates the age-dependent pathological tau accumulation, restores synaptic proteins, and rescues cognitive decline with minimal effects on Ab pathology. In primary neuron and astrocyte coculture, naturally secreted A beta species significantly downregulate GLT-1 steady-state and expression levels. Taken together, our data strongly suggest that GLT-1 restoration is neuroprotective and A beta-induced astrocyte dysfunction represented by a functional loss of GLT-1 may serve as one of the major pathological links between Ab and tau patholog

The role of glial glutamate transporter and its impact on amyloid-beta and tau pathology in Alzheimer’s disease

Alzheimer’s disease (AD) is the leading cause of dementia among elderly in the United States. There is no effective treatment available, in part due to lack of understanding of the full spectrum of the disease mechanisms. Brain is structured in highly heterogeneous and complex way, and the number of different cell lineages, such as neurons, astrocytes, microglia, oligodendrocytes, perivascular macrophages, and capillary endothelial cells, interact with each other and functionally integrate into one highly hierarchical and organized unit. Traditionally, neurodegeneration has been centered for investigation on neurodegenerative diseases like AD. However, recent rapidly growing evidence strongly implicates the pathogenic involvement of non-neuronal cell lineages, such as astrocytes and microglia, and this work may uncover key disease mechanisms. The purpose of this dissertation is to elucidate the role of glutamate transporter 1 (GLT-1) primarily expressed in astrocytes in the progression or initiation of AD pathology. In humans, its loss is evident even in early or prodromal stages of AD and correlates well with cognitive impairment. The loss of GLT-1 causes glutamate dyshomeostasis in the synaptic cleft, which eventually leads to glutamate-induced excitotoxicity and neurodegeneration. While glutamate excitotoxicity has long been a suspected cause of progressive neurodegeneration in AD, it remains unclear whether the loss of the glutamate transporter directly contributes to the pathological buildup of amyloid β (Aβ) and neurofibrillary tau tangles (NFTs) - two key pathological hallmarks of AD. Thus, we hypothesize that the Aβ-induced GLT-1 downregulation links Aβ pathology to tau pathology, synaptic loss, and cognitive decline. To test this hypothesis, we utilized both in vitro and in vivo models of AD and thoroughly performed biochemical and histopathological examinations. We show an age-dependent decrease of GLT-1 in animal models and GLT-1 decrease in human hippocampal samples. In addition, pharmacological restoration of GLT-1 can ameliorate AD-like pathology in an animal model of AD. We also present a possible molecular mechanism that regulates GLT-1 independent of Aβ. The contribution of this research includes a better understanding on the role of astrocyte dysfunction in the early stages of AD and its consequences on the progression of the disease

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

Effective clearance of neurotoxic amyloid-beta (Aβ) from the brain is a critical process to prevent Alzheimer's disease (AD). One major clearance mechanism is Aβ 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 Aβ clearance. Recently, we demonstrated that pro-inflammatory cytokines IL-1β, 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-1β 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-1β. 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-1β. Synthetic microRNA-205-5p, -200b-3p, and -200c-3p mimics recapitulated LRP1 loss in MVECs without IL-1β, and their synthetic antagomirs effectively reversed IL-1β-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-1β-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-1β 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

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

The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress.

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration

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