Human immunodeficiency virus (HIV) infection induces neuronal injuries, with almost 50% of infected individuals developing HIV-associated neurocognitive disorders (HAND). Although highly activate antiretroviral therapy (HAART) has significantly reduced the incidence of severe dementia, the overall prevalence of HAND remains high. Synaptic degeneration is emerging as one of the most relevant neuropathologies associate with HAND. Previous studies have reported critical roles of viral proteins and inflammatory responses in this pathogenesis. Infected cells, including macrophages, microglia and astrocytes, may release viral proteins and other neurotoxins to stimulate neurons and cause excessive calcium influx, overproduction of free radicals and disruption of neurotransmitter hemostasis. The dysregulation of neural circuits likely leads to synaptic damage and loss. Identification of the specific mechanism of the synaptic degeneration may facilitate the development of effective therapeutic approaches to treat HAND.
and coverslips were applied with ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Immunofluorescence was captured using a Zeiss LSM 510 laser-scanning confocal microscope. Statistics. Survival curve comparisons were performed using GraphPad Prism software, which uses the log-rank test. Values for viral burden, cytokine production, and antibody and T cell response experiments are presented as the mean ± SEM. P values for these experiments were calculated with an unpaired, 2-tailed Student's t test. Statistical significance was accepted at a P value of less than 0.05. Study approval. All experiments were performed in compliance with and under the approval of the IACUC of UTMB.
HIV-1 infection of the nervous system causes various neurological diseases, and synaptic degeneration is likely a critical step in the neuropathogenesis. Our prior studies revealed a significant decrease of synaptic protein, specifically in the spinal dorsal horn of patients with HIV-1 in whom pain developed, suggesting a potential contribution of synaptic degeneration to the pathogenesis of HIV-associated pain. However, the mechanism by which HIV-1 causes the spinal synaptic degeneration is unclear. Here, we identified a critical role of microglia in the synaptic degeneration. In primary cortical cultures (day in vitro 14) and spinal cords of 3-to 5-month-old mice (both sexes), microglial ablation inhibited gp120-induced synapse decrease. Fractalkine (FKN), a microglia activation chemokine specifically expressed in neurons, was upregulated by gp120, and knockout of the FKN receptor CX3CR1, which is predominantly expressed in microglia, protected synapses from gp120-induced toxicity. These results indicate that the neuron-to-microglia intercellular FKN/ CX3CR1 signaling plays a role in gp120-induced synaptic degeneration. To elucidate the mechanism controlling this intercellular signaling, we tested the role of the Wnt/-catenin pathway in regulating FKN expression. Inhibition of Wnt/-catenin signaling blocked both gp120-induced FKN upregulation and synaptic degeneration, and gp120 stimulated Wnt/-catenin-regulated FKN expression via NMDA receptors (NMDARs). Furthermore, NMDAR antagonist APV, Wnt/-catenin signaling suppressor DKK1, or knockout of CX3CR1 alleviated gp120-induced mechanical allodynia in mice, suggesting a critical contribution of the Wnt/-catenin/FKN/CX3R1 pathway to gp120-induced pain. These findings collectively suggest that HIV-1 gp120 induces synaptic degeneration in the spinal pain neural circuit by activating microglia via Wnt3a/-catenin-regulated FKN expression in neurons.Synaptic degeneration develops in the spinal cord dorsal horn of HIV patients with chronic pain, but the patients without the pain disorder do not show this neuropathology, indicating a pathogenic contribution of the synaptic degeneration to the development of HIV-associated pain. However, the mechanism underlying the synaptic degeneration is unclear. We report here that HIV-1 gp120, a neurotoxic protein that is specifically associated with the manifestation of pain in HIV patients, induces synapse loss via microglia. Further studies elucidate that gp120 activates microglia by stimulating Wnt/-catenin-regulated fractalkine in neuron. The results demonstrate a critical role of microglia in the pathogenesis of HIV-associated synaptic degeneration in the spinal pain neural circuit.
While astrocytes have been traditionally described as passive supportive cells, studies during the last decade have shown they are active players in many aspects of cnS physiology and function both in normal and disease states. However, the precise mechanisms regulating astrocytes function and interactions within the cnS are still poorly understood. this knowledge gap is due in large part to the limitations of current image analysis tools that cannot process astrocyte images efficiently and to the lack of methods capable of quantifying their complex morphological characteristics. to provide an unbiased and accurate framework for the quantitative analysis of fluorescent images of astrocytes, we introduce a new automated image processing pipeline whose main novelties include an innovative module for cell detection based on multiscale directional filters and a segmentation routine that leverages deep learning and sparse representations to reduce the need of training data and improve performance. extensive numerical tests show that our method performs very competitively with respect to state-of-the-art methods also in challenging images where astrocytes are clustered together. Our code is released open source and freely available to the scientific community.The human brain is a complex network of over 2 × 10 11 neural cells comprising neurons and glial cells. Neuroglia comprise astrocytes, oligodendrocytes, NG2 glia, microglia and all peripheral glia 1 . Astrocytes, a subtype of glial cells with a complex star-shaped morphology, are the most abundant cells in most part of the human brain 2 . Although they were long characterized as supportive cells only involved in maintaining neuron homeostasis and survival, a number of studies during the last decade have revealed that astrocytes play an active role in fundamental brain processes underlying neuronal development and function. Several studies have implicated astrocytes in controlling the development of the nervous system through axon guidance and synaptogenesis. During neural circuit development, astrocytes are responsible for the regularization of the neuronal network by pruning abnormal or dysfunctioning synapses 3 . In addition, together with the pre-and post-synaptic parts of two neuronal synapses, astrocytes can form a so-called "tripartite" synapse that helps modulate synaptic transmission via the release of neurotransmitters such as glutamate and ATP 4,5 . The critical roles of astrocytes in neuronal development and connectivity make them among the most promising targets for innovative therapies designed to treat a range of brain disorders or neurological injuries 6 . As a result, the attention on astrocytes has dramatically increased in recent years.Astrocytes have been shown to reflect their diverse abilities and functions on their special structural design, and alterations in astrocyte morphology are known to correlate to traumatic brain injury, infection, ischemia, autoimmune responses, and neurodegenerative diseases 5,7,8 . For instance, their intricate ar...
Microglia are heterogeneous and ubiquitous CNS-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single cell RNA-seq (scRNA-seq) analysis to compare the transcriptomes of 32760 microglia in adult mouse (C57/Bl) brains and spinal cords to identify microglial subtypes in these CNS compartments. Cortical microglia from 2-month mice consisted of a predominant population of the homeostatic subtype (HOM-M) and a small population (4%) of the inflammatory subtype (IFLAM-M), while spinal microglia consisted of 55% HOM-M and 45% IFLAM-M subtype. Comparison of cortical and spinal microglia at 2, 4 and 8 months revealed consistently a higher composition of the IFLAM-M subtype in the spinal cord. At 8-month, cortical microglia differentiated a small new subtype with interferon response phenotypes (INF-M), while spinal microglia polarized toward a proinflammatory phenotype, as indicated by the increase of microglia expressing IL-1β. To further characterize the differential plasticity of cortical and spinal microglial heterogeneity, we determined the microglial transcriptomes from HIV-1 gp120 transgenic (Tg) mice, a model of HIV-associated neurological disorders. Compared with wilt-type (Wt) cortical microglia, the gp120tg cortical microglia had three new subtypes, with signatures of interferon I response (INF-M), cell proliferation (PLF-M), and myelination or demyelination (MYE-M) respectively; while INF-M and PLF-M subtypes presented at all ages, the MYE-M only at 4-month.In contrast, only the INF-M subtype was observed in the spinal microglia from 2-and 4-month gp120tg mice. Bioinformatic analysis of regulated molecular pathways of individual microglial subtypes indicated that gp120 more severely impaired the biological function of microglia in cortices than in the spinal cord. The results collectively reveal differential heterogeneity and plasticity of cortical and spinal microglia, and suggest functional differentiation of microglia in different CNS compartments.
Microglia have been implicated in neuroinflammatory diseases, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). We demonstrate that microglia mediate EAE disease progression via a mechanism relying on the noncanonical nuclear factor kB (NF-B) pathway. Microglia-specific deletion of the noncanonical NF-B-inducing kinase (NIK) impairs EAE disease progression. Although microglial NIK is dispensable for the initial phase of T cell infiltration into the central nervous system (CNS) and EAE disease onset, it is critical for the subsequent CNS recruitment of inflammatory T cells and monocytes. Our data suggest that following their initial CNS infiltration, T cells activate the microglial noncanonical NF-B pathway, which synergizes with the T cell-derived cytokine granulocyte-macrophage colony-stimulating factor to induce expression of chemokines involved in the second-wave of T cell recruitment and disease progression. These findings highlight a mechanism of microglial function that is dependent on NIK signaling and required for EAE disease progression.
Mammalian target of rapamycin (mTOR) signaling plays a critical role in the regulation of activity-dependent protein synthesis in neurons. It is well established that the GTPase-activating protein tuberous sclerosis complex proteins (2TSC2) is an upstream inhibitor of mTOR. In this study, we show that glutamate stimulation down-regulates TSC2 protein in cortical cultures via NMDA receptor (NMDAR) activation. Interestingly, the mTOR-specific inhibitor rapamycin blocks the glutamate-induced TSC2 down-regulation. This finding suggests that NMDAR activation evokes an mTOR-mediated negative regulation of TSC2. In addition, we also show that the glutamate-induced down-regulation of TSC2 protein is blocked by proteasome inhibitor MG132, indicating the involvement of proteasome-mediated protein degradation. We propose that the NMDAR activation stimulates an mTOR-proteasome pathway to degrade TSC2 protein.
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