RESUMEN
Based on emerging evidence on the role for specific single-nucleotide variants (SNVs) in EIF2AK3 encoding the integrated stress response kinase PERK, in neurodegeneration, we assessed the association of EIF2AK3 SNVs with neurocognitive performance in people with HIV (PWH) using a candidate gene approach. This retrospective study included the CHARTER cohort participants, excluding those with severe neuropsychiatric comorbidities. Genome-wide data previously obtained for 1047 participants and targeted sequencing of 992 participants with available genomic DNA were utilized to interrogate the association of three noncoding and three coding EIF2AK3 SNVs with the continuous global deficit score (GDS) and global neurocognitive impairment (NCI; GDS ≥ 0.5) using univariable and multivariable methods, with demographic, disease-associated, and treatment characteristics as covariates. The cohort characteristics were as follows: median age, 43.1 years; females, 22.8%; European ancestry, 41%; median CD4 + T cell counts, 175/µL (nadir) and 428/µL (current). At first assessment, 70.5% used ART and 68.3% of these had plasma HIV RNA levels ≤ 200 copies/mL. All three noncoding EIF2AK3 SNVs were associated with GDS and NCI (all p < 0.05). Additionally, 30.9%, 30.9%, and 41.2% of participants had at least one risk allele for the coding SNVs rs1805165 (G), rs867529 (G), and rs13045 (A), respectively. Homozygosity for all three coding SNVs was associated with significantly worse GDS (p < 0.001) and more NCI (p < 0.001). By multivariable analysis, the rs13045 A risk allele, current ART use, and Beck Depression Inventory-II value > 13 were independently associated with GDS and NCI (p < 0.001) whereas the other two coding SNVs did not significantly correlate with GDS or NCI after including rs13045 in the model. The coding EIF2AK3 SNVs were associated with worse performance in executive functioning, motor functioning, learning, and verbal fluency. Coding and non-coding SNVs of EIF2AK3 were associated with global NC and domain-specific performance. The effects were small-to-medium in size but present in multivariable analyses, raising the possibility of specific SNVs in EIF2AK3 as an important component of genetic vulnerability to neurocognitive complications in PWH.
Asunto(s)
Infecciones por VIH , Polimorfismo de Nucleótido Simple , eIF-2 Quinasa , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Disfunción Cognitiva/genética , Estudios de Cohortes , eIF-2 Quinasa/genética , Infecciones por VIH/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/psicología , Polimorfismo de Nucleótido Simple/genética , Estudios RetrospectivosRESUMEN
An enhanced understanding of neurotransmitter systems contributing to pain transmission aids in drug development, while the identification of biological variables like age and sex helps in the development of personalized pain management and effective clinical trial design. This study identified enhanced expression of purinergic signaling components specifically in painful inflammation, with levels increased more in women as compared to men. Inflammatory dental pain is common and potentially debilitating; as inflammation of the dental pulp can occur with or without pain, it provides a powerful model to examine distinct pain pathways in humans. In control tissues, P2X3 and P2X2 receptors colocalized with PGP9.5-positive nerves. Expression of the ecto-nucleotidase NTPDase1 (CD39) increased with exposure to extracellular adenosine triphosphate (ATP), implying CD39 acted as a marker for sustained elevation of extracellular ATP. Both immunohistochemistry and immunoblots showed P2X2, P2X3, and CD39 increased in symptomatic pulpitis, suggesting receptors and the ATP agonist were elevated in patients with increased pain. The increased expression of P2X3 and CD39 was more frequently observed in women than men. In summary, this study identifies CD39 as a marker for chronic elevation of extracellular ATP in fixed human tissue. It supports a role for increased purinergic signaling in humans with inflammatory dental pain and suggests the contribution of purines shows sexual dimorphism. This highlights the potential for P2X antagonists to treat pain in humans and stresses the need to consider sex in clinical trials that target pain and purinergic pathways. PERSPECTIVE: This article demonstrates an elevation of ATP-marker CD39 and of ATP receptors P2X2 and P2X3 with inflammatory pain and suggests the rise is greater in women. This highlights the potential for P2X antagonists to treat pain and stresses the consideration of sexual dimorphism in studies of purines and pain.
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Pulpa Dental , Dolor , Masculino , Humanos , Femenino , Pulpa Dental/metabolismo , Inflamación/metabolismo , Adenosina Trifosfato/metabolismo , PurinasRESUMEN
Copper is an essential metal nutrient for life that often relies on redox cycling between Cu(I) and Cu(II) oxidation states to fulfill its physiological roles, but alterations in cellular redox status can lead to imbalances in copper homeostasis that contribute to cancer and other metalloplasias with metal-dependent disease vulnerabilities. Copper-responsive fluorescent probes offer powerful tools to study labile copper pools, but most of these reagents target Cu(I), with limited methods for monitoring Cu(II) owing to its potent fluorescence quenching properties. Here, we report an activity-based sensing strategy for turn-on, oxidation state-specific detection of Cu(II) through metal-directed acyl imidazole chemistry. Cu(II) binding to a metal and oxidation state-specific receptor that accommodates the harder Lewis acidity of Cu(II) relative to Cu(I) activates the pendant dye for reaction with proximal biological nucleophiles and concomitant metal ion release, thus avoiding fluorescence quenching. Copper-directed acyl imidazole 649 for Cu(II) (CD649.2) provides foundational information on the existence and regulation of labile Cu(II) pools, including identifying divalent metal transporter 1 (DMT1) as a Cu(II) importer, labile Cu(II) increases in response to oxidative stress induced by depleting total glutathione levels, and reciprocal increases in labile Cu(II) accompanied by decreases in labile Cu(I) induced by oncogenic mutations that promote oxidative stress.
Asunto(s)
Cobre , Colorantes Fluorescentes , Cobre/metabolismo , Colorantes Fluorescentes/química , Glutatión/metabolismo , Imidazoles , Oncogenes , Oxidación-ReducciónRESUMEN
HIV-associated neurocognitive disorders (HAND) affect over half of HIV-infected individuals, despite antiretroviral therapy (ART). Therapeutically targetable mechanisms underlying HAND remain elusive, partly due to a lack of a representative model. We developed a human-induced pluripotent stem cell (hiPSC)-based model, independently differentiating hiPSCs into neurons, astrocytes, and microglia, and systematically combining to generate a tri-culture with or without HIV infection and ART. Single-cell RNA sequencing analysis on tri-cultures with HIV-infected microglia revealed inflammatory signatures in the microglia and EIF2 signaling in all three cell types. Treatment with the antiretroviral compound efavirenz (EFZ) mostly resolved these signatures. However, EFZ increased RhoGDI and CD40 signaling in the HIV-infected microglia. This activation was associated with a persistent increase in transforming growth factor α production by microglia. This work establishes a tri-culture that recapitulates key features of HIV infection in the CNS and provides a new model to examine the effects of infection, its treatment, and other co-morbid conditions.
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Factor 2 Eucariótico de Iniciación/metabolismo , Infecciones por VIH/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Microglía/virología , Neuronas/virología , Alquinos/farmacología , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa , Astrocitos/metabolismo , Astrocitos/virología , Benzoxazinas/farmacología , Antígenos CD40/metabolismo , Diferenciación Celular , Células Cultivadas , Ciclopropanos/farmacología , Citocinas/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , Humanos , Células Madre Pluripotentes Inducidas/virología , Inflamación/metabolismo , Inflamación/virología , Microglía/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Factor de Crecimiento Transformador alfa/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismoRESUMEN
E2F1 is a transcription factor classically known to regulate G0/G1 to S phase progression in the cell cycle. In addition, E2F1 also regulates a wide range of apoptotic genes and thus has been well studied in the context of neuronal death and neurodegenerative diseases. However, its function and regulation in the mature central nervous system are not well understood. Alternative splicing is a well-conserved post-transcriptional mechanism common in cells of the CNS and is necessary to generate diverse functional modifications to RNA or protein products from genes. Heretofore, physiologically significant alternatively spliced E2F1 transcripts have not been reported. In the present study, we report the identification of two novel alternatively spliced E2F1 transcripts: E2F1b, an E2F1 transcript retaining intron 5, and E2F1c, an E2F1 transcript excluding exon 6. These alternatively spliced transcripts are observed in the brain and neural cell types including neurons, astrocytes, and undifferentiated oligodendrocytes. The expression of these E2F1 transcripts is distinct during maturation of primary hippocampal neuroglial cells. Pharmacologically-induced global translation inhibition with cycloheximide, anisomycin or thapsigargin lead to significantly reduced expression of E2F1a, E2F1b and E2F1c. Conversely, increasing neuronal activity by elevating the concentration of potassium chloride selectively increased the expression of E2F1b. Furthermore, experiments expressing these variants in vitro show the transcripts can be translated to generate a protein product. Taken together, our data suggest that the alternatively spliced E2F1 transcript behave differently than the E2F1a transcript, and our results provide a foundation for future investigation of the function of E2F1 splice variants in the CNS.
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Empalme Alternativo , Factor de Transcripción E2F1/genética , Hipocampo/metabolismo , Animales , Células Cultivadas , Factor de Transcripción E2F1/metabolismo , Hipocampo/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
HIV-associated neurocognitive disorders (HANDs) share common symptoms with Alzheimer's disease (AD), which is characterized by amyloid-ß (Aß) plaques. Plaques are formed by aggregation of Aß oligomers, which may be the toxic species in AD pathogenesis, and oligomers are generated by cleavage of amyloid precursor protein (APP) by ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD. Although studies have also found evidence of altered APP processing in HIV+ patients, it is unknown whether increased BACE1 expression or Aß oligomer production is a common neuropathological feature of HAND. Moreover, it is unknown whether BACE1 or APP is involved in the excitotoxic, NMDAR-dependent component of HIV-associated neurotoxicity in vitro Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDAR-dependent elevation of BACE1 and subsequent altered processing of APP. Supporting this, we observed elevated levels of BACE1 and Aß oligomers in CNS of male and female HIV+ patients. In a model of HIV-associated neurotoxicity in which rat neurons are treated with supernatants from HIV-infected human monocyte-derived macrophages, we observed NMDAR-dependent elevation of BACE1 protein. NMDA treatment also increased BACE1 and both pharmacological BACE1 inhibition and genetic loss of APP were partially neuroprotective. Moreover, in APP knock-out (APP-/-) mouse neurons, NMDA-induced toxicity was BACE1 independent, indicating that cytotoxicity of BACE1 is dependent upon APP cleavage. Our findings suggest that increased BACE1 and the resultant Aß oligomer production may contribute to HIV-associated neuropathogenesis and inhibition of BACE1 could have therapeutic potential in HANDs.SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HANDs) represent a range of cognitive impairments affecting â¼50% of HIV+ individuals. The specific causes of HAND are unknown, but evidence suggests that HIV-infected macrophage infiltration into the brain may cause neuronal damage. Herein, we show that neurons treated with conditioned media from HIV-infected macrophages have increased expression of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein implicated in Alzheimer's disease pathogenesis. Moreover, inhibition of BACE1 prevented neuronal loss after conditioned media exposure, but had no effect on HIV-associated neurotoxicity in neurons lacking its cleavage target amyloid precursor protein. We also observed increased BACE1 expression in HIV+ patient brain tissue, confirming the potential relevance of BACE1 as a therapeutic target in HANDs.
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Complejo SIDA Demencia/genética , Complejo SIDA Demencia/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Infecciones por VIH/patología , Neuronas/patología , Adulto , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Femenino , Hipocampo/metabolismo , Humanos , Macrófagos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , N-Metilaspartato/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
BACKGROUND: Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. METHODS: We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet's post hoc tests. RESULTS: We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1ß in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. CONCLUSION: This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation.
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Antiinflamatorios/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Butileno Glicoles/farmacología , Endotelio Vascular/efectos de los fármacos , Glucósidos/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Microvasos/efectos de los fármacos , Animales , Barrera Hematoencefálica/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Microvasos/metabolismoRESUMEN
MDMx/MDM4 is a negative regulator of the p53 tumor suppressor protein and is necessary for survival in dividing cells. MDMx is also expressed in postmitotic neurons, with prosurvival roles that are independent of its extensively described roles in carcinogenesis. We and others have shown a role for MDMx loss in neuronal death in vitro and in vivo in several neurodegenerative diseases. Further, we have recently shown that MDMx is targeted for proteolytic degradation by calcium-dependent proteases, calpains, in neurons in vitro, and that MDMx overexpression provided partial neuroprotection in a model of HIV-associated neurodegeneration. Here, we assessed whether amyloid ß (Aß)-induced MDMx degradation occurred in Alzheimer's Disease (AD) models. Our data shows an age-dependent reduction in MDMx levels in cholinergic neurons within the cortex of adult mice expressing the swedish mutant of the amyloid precursor protein, APP in the Tg2576 murine model of AD. In vitro, Aß treatment of primary cortical neurons led to the caspase-dependent MDMx degradation. Our findings suggest that MDMx degradation associated with neuronal death occurs via caspase activation in neurons, and that the progressive loss of MDMx protein represents a potential mechanism of Aß-induced neuronal death during disease progression in AD.
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Caspasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Envejecimiento/metabolismo , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Femenino , Ratones , Ratones Mutantes , Degeneración Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/farmacologíaRESUMEN
The transcription factor E2F1 activates gene targets required for G1 -S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson's Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in N-methyl-d-aspartate (NMDA) receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. In addition, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in post-mitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HIV-associated neurocognitive disorders pathogenesis as well as several other diseases of the CNS. After crossing the blood-brain barrier, HIV-infected monocytes differentiate into macrophages and release excitotoxins and inflammatory factors including glutamate into the brain parenchyma (1). These factors stimulate neuronal N-Methyl-d-aspartate (NMDA) receptors (2), causing calcium influx (3) and subsequent activation of the cysteine protease calpain (4). Activated calpain cleaves multiple substrates including E2F1, producing a stabilized protein fragment with truncations at the N- and C-terminus (5). Calpain-cleaved E2F1 may contribute to calpain-mediated neuronal damage observed in NMDA receptor-mediated neurotoxicity (6).
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Calpaína/metabolismo , Factor de Transcripción E2F1/metabolismo , Infecciones por VIH/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/virología , Ratas , Ratas Sprague-DawleyRESUMEN
Heme oxygenase-1 (HO-1) is an inducible, detoxifying enzyme that is critical for limiting oxidative stress, inflammation, and cellular injury within the CNS and other tissues. Here, we demonstrate a deficiency of HO-1 expression in the brains of HIV-infected individuals. This HO-1 deficiency correlated with cognitive dysfunction, HIV replication in the CNS, and neuroimmune activation. In vitro analysis of HO-1 expression in HIV-infected macrophages, a primary CNS HIV reservoir along with microglia, demonstrated a decrease in HO-1 as HIV replication increased. HO-1 deficiency correlated with increased culture supernatant glutamate and neurotoxicity, suggesting a link among HIV infection, macrophage HO-1 deficiency, and neurodegeneration. HO-1 siRNA knockdown and HO enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with nonselective pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. Furthermore, IFN-γ, which is increased in CNS HIV infection, reduced HO-1 expression in cultured human astrocytes and macrophages. These findings indicate that HO-1 is a protective host factor against HIV-mediated neurodegeneration and suggest that HO-1 deficiency contributes to this degeneration. Furthermore, these results suggest that HO-1 induction in the CNS of HIV-infected patients on antiretroviral therapy could potentially protect against neurodegeneration and associated cognitive dysfunction.
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Infecciones por VIH/fisiopatología , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/fisiología , Enfermedades del Sistema Nervioso/fisiopatología , Adulto , Anciano , Antioxidantes/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Sistema Nervioso Central , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/virología , Estudios de Cohortes , Dimetilfumarato , Femenino , Fumaratos/química , Infecciones por VIH/metabolismo , VIH-1 , Humanos , Inflamación , Modelos Lineales , Macrófagos/metabolismo , Macrófagos/virología , Masculino , Microglía/metabolismo , Persona de Mediana Edad , Enfermedades del Sistema Nervioso/metabolismo , Estrés Oxidativo , Corteza Prefrontal/patología , ARN Interferente Pequeño/metabolismo , Replicación ViralRESUMEN
Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often up-regulated and activated in models of neuronal death. However, despite its well-studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In this study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age-dependent olfactory and memory-related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age-dependent loss of post-synaptic protein-95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages, in which behavioral and synaptic perturbations were observed. Finally, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age-dependent behavioral deficits and synaptic perturbations. E2F1 is a transcription factor regulating cell cycle progression and apoptosis. Although E2F1 dysregulation under toxic conditions can lead to neuronal death, little is known about its physiologic activity in the healthy brain. Here, we report significant age-dependent olfactory and memory deficits in mice with dysfunctional E2F1. Coincident with these behavioral changes, we also found age-matched synaptic disruption and persisting reduction in adult neurogenesis. Our study demonstrates that E2F1 contributes to physiologic brain structure and function.
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Envejecimiento/genética , Envejecimiento/psicología , Conducta Animal/fisiología , Factor de Transcripción E2F1/genética , Mutación/genética , Sinapsis/patología , Animales , Western Blotting , Células Cultivadas , Marcación de Gen , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Memoria/fisiología , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Odorantes , Trastornos del Olfato/genética , Trastornos del Olfato/psicología , Equilibrio Postural/genética , Equilibrio Postural/fisiología , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-Dawley , Reconocimiento en Psicología , Olfato/genética , Olfato/fisiología , Sinaptosomas/fisiologíaRESUMEN
Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.
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Complejo SIDA Demencia/metabolismo , Calpaína/metabolismo , Lóbulo Frontal/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Complejo SIDA Demencia/inmunología , Complejo SIDA Demencia/patología , Animales , Proteínas de Ciclo Celular , Muerte Celular , Células Cultivadas , Medios de Cultivo Condicionados/toxicidad , Lóbulo Frontal/patología , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/virología , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Proteínas Nucleares/genética , Proteolisis , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Despite antiretroviral therapy (ART), HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation and neurotoxin release from infected and/or activated macrophages/microglia. Furthermore, inflammation and immune activation within both the CNS and periphery correlate with disease progression and morbidity in ART-treated individuals. Accordingly, drugs targeting these pathological processes in the CNS and systemic compartments are needed for effective, adjunctive therapy. Using our in vitro model of HIV-mediated neurotoxicity, in which HIV-infected monocyte-derived macrophages release excitatory neurotoxins, we show that HIV infection dysregulates the macrophage antioxidant response and reduces levels of heme oxygenase-1 (HO-1). Furthermore, restoration of HO-1 expression in HIV-infected monocyte-derived macrophages reduces neurotoxin release without altering HIV replication. Given these novel observations, we have identified dimethyl fumarate (DMF), used to treat psoriasis and showing promising results in clinical trials for multiple sclerosis, as a potential neuroprotectant and HIV disease-modifying agent. DMF, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and neurotoxin release. Two distinct mechanisms are proposed: inhibition of NF-κB nuclear translocation and signaling, which could contribute to the suppression of HIV replication, and induction of HO-1, which is associated with decreased neurotoxin release. Finally, we found that DMF attenuates CCL2-induced monocyte chemotaxis, suggesting that DMF could decrease recruitment of activated monocytes to the CNS in response to inflammatory mediators. We propose that dysregulation of the antioxidant response during HIV infection drives macrophage-mediated neurotoxicity and that DMF could serve as an adjunctive neuroprotectant and HIV disease modifier in ART-treated individuals.
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Fármacos Anti-VIH/farmacología , Antioxidantes/metabolismo , Fumaratos/farmacología , Inmunosupresores/farmacología , Macrófagos/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Replicación Viral/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/inmunología , Animales , Antioxidantes/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/inmunología , Corteza Cerebral/patología , Dimetilfumarato , VIH-1/efectos de los fármacos , VIH-1/inmunología , Humanos , Macrófagos/citología , Macrófagos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/patología , Neuronas/patología , Neuronas/virología , Ratas , Ratas Sprague-Dawley , Replicación Viral/inmunologíaRESUMEN
Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP(3) ), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.
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Péptidos y Proteínas de Señalización Intercelular/farmacología , Fármacos Neuroprotectores , Neurotoxinas/toxicidad , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Apelina , Western Blotting , Encéfalo/citología , Calcio/metabolismo , Calpaína/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fenómenos Electrofisiológicos , Infecciones por VIH/patología , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Macrófagos/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Malignant rhabdoid tumors (MRTs) are aggressive tumors associated with mutations in the SMARCB1 gene. In experimental systems, the loss of SMARCB1 is hypothesized to alter p16(INK4A) pathways resulting in the repression of tumor suppressors. To determine whether these pathways are deregulated in human MRT, we used immunohistochemistry on tissue microarrays to evaluate p16(INK4A)/E2F1/RB and p14(ARF)/MDM2/p53 pathways in 25 atypical teratoid/rhabdoid tumors (AT/RT) and 11 non-CNS MRT. p16(INK4A) was negative or showed focal weak expression. p16(INK4A) downstream targets CDK4/cyclin D1/ppRB were variably expressed at moderate to low levels; E2F1 was negative. Unexpectedly, p14(ARF) expression was seen in many cases, which correlated positively with p53 and inversely with MDM2 immunostaining in AT/RT. TP53 mutational analysis in 19 of 25 AT/RT and in 8 of 11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by p14(ARF). Finally, nucleophosmin, a protein that stabilizes p53, was positive in most cases and colocalized with p53. Together, these data suggest that, in MRT, there is deregulation not only of p16(INK4A) but also of the p14(ARF) pathway. These results provide insights into cell cycle deregulation in the pathogenesis of human MRT and may aid in the design and evaluation of potential therapies for these tumors.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Transducción de Señal/fisiología , Teratoma/fisiopatología , Proteína p14ARF Supresora de Tumor/metabolismo , Preescolar , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Lactante , Masculino , Análisis por Micromatrices , Proteína SMARCB1 , Índice de Severidad de la Enfermedad , Transducción de Señal/genética , Estadísticas no Paramétricas , Teratoma/genética , Teratoma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.
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Complejo SIDA Demencia/patología , Potencial de la Membrana Mitocondrial/fisiología , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/virología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Neuronas/virología , Complejo SIDA Demencia/metabolismo , Animales , Calpaína/antagonistas & inhibidores , Calpaína/fisiología , Técnicas de Cultivo de Célula/métodos , Muerte Celular/fisiología , Células Cultivadas , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/virología , Proteínas Asociadas a Microtúbulos/deficiencia , Degeneración Nerviosa/virología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , RodaminasRESUMEN
As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.
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Complejo SIDA Demencia/metabolismo , Citoplasma/metabolismo , Factor de Transcripción E2F1/metabolismo , VIH/fisiología , Neuronas/metabolismo , Complejo SIDA Demencia/patología , Animales , Apoptosis , Caspasas/biosíntesis , Caspasas/genética , Proliferación Celular , Células Cultivadas , Corteza Cerebral/metabolismo , Ciclina A/biosíntesis , Ciclina A/genética , Factor de Transcripción E2F1/genética , Humanos , Macrófagos/virología , Neuronas/patología , Neuronas/virología , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Ratas , Ratas Sprague-Dawley , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/genética , Activación Transcripcional , Proteína p14ARF Supresora de Tumor/biosíntesis , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
As organisms designed to depend upon oxygen to sustain life, humans are necessarily and continually exposed to damaging oxidizing agents. As a vital protective measure, oxygen-dependent organisms have developed a highly evolutionarily conserved mechanism for preventing oxidative stress. NF-E2 (nuclear factor (erythroid-derived 2))-related factor-2 (Nrf2) is the primary regulator of this endogenous antioxidant response. Many diseases that plague human society, ranging from various cancers to neurodegenerative diseases, have oxidative stress as a component of their etiology, and thus, much disease research has focused on Nrf2, both as a potential point of biological failure and as a promising therapeutic target. As a transcription factor, Nrf2 is active only when it is nuclear, and is regulated largely by its subcellular distribution. Thus, Nrf2 protein levels and subcellular localization are both key pieces of information when studying the endogenous antioxidant response. Immunofluorescent analysis (IFA) of Nrf2 in human tissue is a particularly powerful tool in the study of Nrf2 in disease, because it allows examination of both of these regulatory mechanisms that modulate Nrf2 activity.
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Antioxidantes/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Factor 2 Relacionado con NF-E2/metabolismo , Anticuerpos , Humanos , Microscopía Confocal , Adhesión en ParafinaRESUMEN
Astrocytes perform vital maintenance, functional enhancement, and protective roles for their associated neurons; however these same mechanisms may become deleterious for neurons under some conditions. In this review, we highlight two normally protective pathways, the endoplasmic reticulum (ER) stress response and an endogenous antioxidant response, which may become neurotoxic when activated in astrocytes during the inflammation associated with neurodegeneration. Stimulation of these multifaceted pathways affects a panoply of cellular processes. Of particular importance is the effect these pathways have on the homeostasis of the excitatory amino acid neurotransmitter, glutamate. The endogenous antioxidant response increases extracellular glutamate in the pursuit of making the cellular antioxidant, glutathione, by increasing expression of the xCT subunit of the cystine/glutamate antiporter. Meanwhile, inflammatory mediators such as TNFα reduce levels of membrane-bound glutamate scavenging proteins such as the excitatory amino acid transporters. Together, these cellular activities may result in a net increase in extracellular glutamate that could alter neuronal function and lead to excitotoxicity. Here we discuss the role of N-methyl-D-aspartate receptors, which, when excessively stimulated by glutamate, can cause neuronal dysfunction and loss via activation of calpains. While there are other pathways acting in concert or parallel to those we describe here, this review explores a rationale to explain how two protective mechanisms may result in neuronal loss during neurodegeneration.
RESUMEN
Neuronal damage in human immunodeficiency virus type 1 (HIV-1) infection in the brain is thought to occur at least in part through NMDA receptor (NMDAR) excitation initiated by soluble neurotoxins from HIV-infected brain macrophages. Furthermore, brain regions enriched in NMDAR-2A (NR2A) and NMDAR-2B (NR2B) subunits, such as the hippocampus, are particularly vulnerable. Using cultured rat hippocampal cells and HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we examined the role of NR2A and NR2B in HIV/MDM-induced hippocampal neuronal death. We used the primary HIV-1 strain Jago derived from the CSF of an individual with HIV-associated dementia and that robustly replicates in MDM. We found the following: (1) hippocampal neuronal susceptibility to HIV/MDM excitotoxins varies according to the developmental expression patterns of NR2A and NR2B; (2) NMDAR activation by HIV/MDM results in neuronal calpain activation, which results in neuronal death; and (3) selective antagonists of homomeric NR2B/NR2B- and heteromeric NR2A/NR2B-containing NMDARs, as well as an inhibitor of calpain activity, afford neuroprotection against HIV/MDM. These studies establish a clear link between macrophage HIV infection, neuronal NR2A and NR2B activation, and calpain-mediated hippocampal neuronal death. They further suggest a dominant role for NR2A and NR2B in determining neuronal susceptibility in HIV-infected brain. Antagonists of NR2A and NR2B subunits as well as inhibitors of calpain activation offer attractive neuroprotective approaches against HIV in both developing and mature brain.