RESUMEN
BACKGROUND: Interleukin-1ß (IL-1ß) is one of the most important cytokine secreted by activated microglia as it orchestrates the vicious cycle of inflammation by inducing the expression of various other pro-inflammatory cytokines along with its own production. Microglia-mediated IL-1ß production is a tightly regulated mechanism which involves the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome pathway. Our previous study suggests the critical role of heat shock protein 60 (HSP60) in IL-1ß-induced inflammation in microglia through TLR4-p38 MAPK axis. However, whether HSP60 regulates endogenous IL-1ß production is not known. Therefore, to probe the underlying mechanism, we elucidate the role of HSP60 in endogenous IL-1ß production. METHODS: We used in vitro (N9 murine microglial cells) and in vivo (BALB/c mouse) models for our study. HSP60 overexpression and knockdown experiment was done to elucidate the role of HSP60 in endogenous IL-1ß production by microglia. Western blotting and quantitative real-time PCR was performed using N9 cells and BALB/c mice brain, to analyze various proteins and transcript levels. Reactive oxygen species levels and mitochondrial membrane depolarization in N9 cells were analyzed by flow cytometry. We also performed caspase-1 activity assay and enzyme-linked immunosorbent assay to assess caspase-1 activity and IL-1ß production, respectively. RESULTS: HSP60 induces the phosphorylation and nuclear localization of NF-κB both in vitro and in vivo. It also induces perturbation in mitochondrial membrane potential and enhances reactive oxygen species (ROS) generation in microglia. HSP60 further activates NLRP3 inflammasome by elevating NLRP3 expression both at RNA and protein levels. Furthermore, HSP60 enhances caspase-1 activity and increases IL-1ß secretion by microglia. Knockdown of HSP60 reduces the IL-1ß-induced production of IL-1ß both in vitro and in vivo. Also, we have shown for the first time that knockdown of HSP60 leads to decreased IL-1ß production during Japanese encephalitis virus (JEV) infection, which eventually leads to decreased inflammation and increased survival of JEV-infected mice. CONCLUSION: HSP60 mediates microglial IL-1ß production by regulating NLRP3 inflammasome pathway and reduction of HSP60 leads to reduction of inflammation in JEV infection.
Asunto(s)
Chaperonina 60/farmacología , Regulación de la Expresión Génica/fisiología , Interleucina-1beta/metabolismo , Microglía/efectos de los fármacos , Proteínas Mitocondriales/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Virus de la Encefalitis Japonesa (Subgrupo)/fisiología , Encefalitis Japonesa/metabolismo , Encefalitis Japonesa/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Morfolinos/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Interferente Pequeño/farmacologíaRESUMEN
Upon publication of the original article [1], it was noticed that there is an error in Fig. 10, the dialog box in panel (b) was missing. The correct Fig. 10 is shown below.
RESUMEN
OBJECTIVE: To present a retrospective study from patients with spinal cysticercosis (SC), diagnosed within the last 30 years in Mexican and Indian neurological referral centers. METHODS: This is a retrospective and comparative study of the clinical and radiological profile between Mexican and Indian patients with spinal neurocysticercosis during a 30-year period and a review of the literature during the same period. RESULTS: Twenty-seven SC patients were included: 19 from Mexico and 8 from India. SC presented predominantly with motor symptoms (21/27 patients): paraparesis and paraplegia were the most common signs; one-third of patients presented sphincter dysfunction. Imaging studies showed that parasites in vesicular stage were more frequent in patients from Mexico, while degenerative stages predominated in India. Association of subarachnoid cysticerci and hydrocephalus was observed only in Mexican patients. CONCLUSIONS: Despite the limitations of this study, the collected information supports the existence of differences in the clinical and radiological traits of SC patients between Asian and Latin-American hospitals. The possible biological factors that may underlie these differences are discussed.
Asunto(s)
Neurocisticercosis/diagnóstico , Enfermedades de la Médula Espinal/diagnóstico , Taenia solium , Adulto , Anciano , Animales , Femenino , Humanos , Hidrocefalia/parasitología , India , Imagen por Resonancia Magnética , Masculino , México , Persona de Mediana Edad , Neurocisticercosis/complicaciones , Derivación y Consulta , Estudios Retrospectivos , Enfermedades de la Médula Espinal/complicacionesRESUMEN
Traumatic brain injury (TBI) contributes to fatalities and neurological disabilities worldwide. While primary injury causes immediate damage, secondary events contribute to long-term neurological defects. Contusions (Ct) are primary injuries correlated with poor clinical prognosis, and can expand leading to delayed neurological deterioration. Pericontusion (PC) (penumbra), the region surrounding Ct, can also expand with edema, increased intracranial pressure, ischemia, and poor clinical outcome. Analysis of Ct and PC can therefore assist in understanding the pathobiology of TBI and its management. This study on human TBI brains noted extensive neuronal, astroglial and inflammatory changes, alterations in mitochondrial, synaptic and oxidative markers, and associated proteomic profile, with distinct differences in Ct and PC. While Ct displayed petechial hemorrhages, thrombosis, inflammation, neuronal pyknosis, and astrogliosis, PC revealed edema, vacuolation of neuropil, axonal loss, and dystrophic changes. Proteomic analysis demonstrated altered immune response, synaptic, and mitochondrial dysfunction, among others, in Ct, while PC displayed altered regulation of neurogenesis and cytoskeletal architecture, among others. TBI brains displayed oxidative damage, glutathione depletion, mitochondrial dysfunction, and loss of synaptic proteins, with these changes being more profound in Ct. We suggest that analysis of markers specific to Ct and PC may be valuable in the evaluation of TBI pathobiology and therapeutics. We have characterized the primary injury in human traumatic brain injury (TBI). Contusions (Ct) - the injury core displayed hemorrhages, inflammation, and astrogliosis, while the surrounding pericontusion (PC) revealed edema, vacuolation, microglial activation, axonal loss, and dystrophy. Proteomic analysis demonstrated altered immune response, synaptic and mitochondrial dysfunction in Ct, and altered regulation of neurogenesis and cytoskeletal architecture in PC. Ct displayed more oxidative damage, mitochondrial, and synaptic dysfunction compared to PC.
Asunto(s)
Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Encéfalo/metabolismo , Encéfalo/patología , Contusiones/metabolismo , Contusiones/patología , Lesiones Encefálicas/genética , Contusiones/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Oxidación-Reducción , Proteómica/métodosRESUMEN
UNLABELLED: MicroRNAs (miRNAs) are single-stranded small RNA molecules that regulate various cellular processes. miRNA 155 (miR-155) regulates various aspects of innate and adaptive immune responses and plays a key role in various viral infections and the resulting neuroinflammation. The present study evaluated the involvement of miR-155 in modulating Japanese encephalitis virus (JEV)-induced neuroinflammation. We observed that miR-155 expression was upregulated during JEV infection of mouse primary microglia, the BV-2 microglia cell line, and in both mouse and human brains. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of the Src homology 2-containing inositol phosphatase 1 (SHIP1) 3' untranslated region (UTR) by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher beta interferon (IFN-ß) and proinflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations, we conclude that miR-155 modulates the neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV-induced neuroinflammation. IMPORTANCE: Japanese encephalitis virus (JEV), a member of the family Flaviviridae that causes Japanese encephalitis (JE), is the most common mosquito-borne encephalitis virus in the Asia-Pacific region. The disease is feared, as currently there are no specific antiviral drugs available. JEV targets the central nervous system, leading to high mortality and neurological and psychiatric sequelae in some of those who survive. The level of inflammation correlates well with the clinical outcome in patients. Recently, microRNA (miRNA), a single-stranded noncoding RNA, has been implicated in various brain disorders. The present study investigates the role of miRNA in JEV-induced neuroinflammation. Our results show that miRNA 155 (miR-155) targets the Src homology 2-containing inositol phosphatase 1 (SHIP1) protein and promotes inflammation by regulating the NF-κB pathway, increasing the expression of various proinflammatory cytokines and the antiviral response. Thus, miR-155 is a potential therapeutic target to develop antivirals in JE and other brain disorders where inflammation plays a significant role in disease progression.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/patología , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Animales , Encéfalo/patología , Células Cultivadas , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Endogámicos BALB C , Microglía/inmunología , Fosfatidilinositol-3,4,5-Trifosfato 5-FosfatasasRESUMEN
Lafora disease (LD) is a teenage-onset inherited progressive myoclonus epilepsy characterized by the accumulations of intracellular inclusions called Lafora bodies and caused by mutations in protein phosphatase laforin or ubiquitin ligase malin. But how the loss of function of either laforin or malin causes disease pathogenesis is poorly understood. Recently, neuronatin was identified as a novel substrate of malin that regulates glycogen synthesis. Here we demonstrate that the level of neuronatin is significantly up-regulated in the skin biopsy sample of LD patients having mutations in both malin and laforin. Neuronatin is highly expressed in human fetal brain with gradual decrease in expression in developing and adult brain. However, in adult brain, neuronatin is predominantly expressed in parvalbumin-positive GABAergic interneurons and localized in their processes. The level of neuronatin is increased and accumulated as insoluble aggregates in the cortical area of LD brain biopsy samples, and there is also a dramatic loss of parvalbumin-positive GABAergic interneurons. Ectopic expression of neuronatin in cultured neuronal cells results in increased intracellular Ca(2+), endoplasmic reticulum stress, proteasomal dysfunction, and cell death that can be partially rescued by malin. These findings suggest that the neuronatin-induced aberrant Ca(2+) signaling and endoplasmic reticulum stress might underlie LD pathogenesis.
Asunto(s)
Señalización del Calcio , Retículo Endoplásmico/metabolismo , Enfermedad de Lafora/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adolescente , Adulto , Factores de Edad , Biopsia/métodos , Encéfalo/patología , Calcio/metabolismo , Proteínas Portadoras/genética , Niño , Humanos , Lactante , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Transducción de Señal , Piel/patología , Transfección , Ubiquitina-Proteína LigasasRESUMEN
Toll-like receptor 7 (TLR7) known to recognize guanidine-rich ssRNA has been shown to mount vital host defense mechanism against many viruses including flaviviruses. Signal transduction through TLR7 has been shown to produce type-1 interferon and proinflammatory mediators, thereby initiating essential innate immune response against ssRNA viruses in hosts. Systemic and brain specific TLR7 knock-down mice (TLR7(KD)) were generated using vivo-morpholinos. These mice were then subcutaneously challenged with lethal dose of JEV (GP78 strain) and were subsequently analyzed for survival. Significant difference in susceptibility to JEV between wild-type and systemic TLR7(KD) mice was observed whereas, no difference in susceptibility to JEV infection was seen in brain-specific TLR7(KD) mice. Significant decreases in IFN-α and antiviral proteins were also observed in both TLR7(KD) mice along with increased viral loads in their brain. Owing to increased viral load, increases in levels of various proinflammatory cyto/chemokines, increased microglial activation and infiltration of peripheral immune cells in brain of TLR7(KD) mice were also observed. Immunocytochemistry and RNA co-immunoprecipitation performed with JEV-infected N2a or HT22 cells indicated endosomal localization and confirmed interaction between JEV ssRNA with TLR7. Treatment of mice with imiquimod, a TLR7 agonist, prior to JEV infection resulted in their increased survival. Overall, our results suggest that the TLR7 response following JEV infection promotes type-1 interferon production and generation of antiviral state which might contribute to protective effect in systemic infection.
Asunto(s)
Encefalitis Japonesa/inmunología , Inmunidad Innata , Glicoproteínas de Membrana/metabolismo , Receptor Toll-Like 7/metabolismo , Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Animales , Encéfalo/inmunología , Encéfalo/virología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/prevención & control , Encefalitis Japonesa/virología , Técnicas de Silenciamiento del Gen , Humanos , Imiquimod , Interferón Tipo I/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Ratones Endogámicos BALB C , Ratones Transgénicos , Microglía/fisiología , Neuronas/inmunología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/genéticaRESUMEN
Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.
Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/aislamiento & purificación , Rabia/diagnóstico , Adulto , Anciano , Animales , Anticuerpos Neutralizantes/sangre , Niño , Femenino , Humanos , Inmunoensayo/métodos , India , Masculino , Persona de Mediana Edad , ARN Viral/genética , Rabia/veterinaria , Adulto JovenRESUMEN
BACKGROUND: Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection. RESULTS: We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism. CONCLUSIONS: Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.
RESUMEN
The frequencies of 10 opportunistic DNA viruses were determined by multiplex real-time PCR in paired cerebrospinal fluid (CSF) and brain tissue of HIV-infected individuals. In the CSF, viruses were detectable in 45/55 cases: JC virus (JCV) in 62%, Epstein-Barr virus (EBV) in 44%, cytomegalovirus (CMV) in 25%, varicella-zoster virus (VZV) in 3.6%, herpes simplex virus 1 (HSV-1) in 1.8%, and human herpesvirus 6 (HHV-6) in 1.8% of cases. A single virus was detectable in 20 cases, 19 cases had coinfection with two viruses, and 6 cases were positive for three viruses. JCV was detectable in the CSF of 62% of cases and in 42% of brain tissues, with higher loads in progressive multifocal leukoencephalopathy (PML) (P < 0.05).
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/mortalidad , Criptococosis/mortalidad , Infecciones por Virus ADN/epidemiología , Virus ADN/aislamiento & purificación , Infecciones por VIH/complicaciones , Toxoplasmosis/mortalidad , Tuberculosis/mortalidad , Infecciones Oportunistas Relacionadas con el SIDA/virología , Encéfalo/virología , Líquido Cefalorraquídeo/virología , Criptococosis/complicaciones , Infecciones por Virus ADN/virología , Virus ADN/clasificación , Virus ADN/genética , Humanos , India/epidemiología , Reacción en Cadena de la Polimerasa Multiplex , Prevalencia , Toxoplasmosis/complicaciones , Tuberculosis/complicacionesRESUMEN
Lafora disease (LD) is an autosomal recessive progressive myoclonic epilepsy characterized by the presence of intracellular polyglucosan inclusions commonly known as Lafora bodies in many tissues, including the brain, liver and skin. The disease is caused by mutations in either EPM2A gene, encoding the protein phosphatase, laforin, or EPM2B gene, encoding the ubiquitin ligase, malin. But how mutations in these two genes cause disease pathogenesis is poorly understood. In this study, we show that the Lafora bodies in the axillary skin and brain stain positively for the ubiquitin, the 20S proteasome and the molecular chaperones Hsp70/Hsc70. Interestingly, mutant malins that are misfolded also frequently colocalizes with Lafora bodies in the skin biopsy sample of the respective LD patient. The expression of disease-causing mutations of malin in Cos-7 cells results in the formation of the profuse cytoplasmic aggregates that colocalize with the Hsp70/Hsc70 chaperones and the 20S proteasome. The mutant malin expressing cells also exhibit proteasomal dysfunction and cell death. Overexpression of Hsp70 decreases the frequency of the mutant malin aggregation and protects from mutant malin-induced cell death. These findings suggest that Lafora bodies consist of abnormal proteins, including mutant malin, targeted by the chaperones or the proteasome for their refolding or clearance, and failure of these quality control systems could lead to LD pathogenesis. Our data also indicate that the Hsp70 chaperone could be a potential therapeutic target of LD.
Asunto(s)
Proteínas Portadoras/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Cuerpos de Inclusión/metabolismo , Enfermedad de Lafora , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Células COS , Muerte Celular , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Glucanos/genética , Proteínas del Choque Térmico HSC70/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Cuerpos de Inclusión/patología , Enfermedad de Lafora/genética , Enfermedad de Lafora/metabolismo , Enfermedad de Lafora/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Neuronal/glioneuronal tumors are uncommon neoplasms of the CNS with frequent association with refractory epilepsy. Reports documenting the entire spectrum of neuronal/glioneuronal tumors are scarce in the literature. Zulch et al. from Germany in a large series reported that neuronal/glioneuronal tumors accounted for 0.4% (38/9000 cases) of all brain tumors, with similar incidence reported from Japan (0.4%), with higher incidence from Korea (2.1%). However, data from the Indian subcontinent are lacking. We reviewed 244 cases of neuronal/glioneuronal tumors of the CNS diagnosed over the last decade at our Institute and they constituted 0.86% of all CNS tumors (244/28061) received in that period. Mean age at presentation was 25.06 years (range: 1-75 years) with male preponderance (M:F = 1.54 : 1). The majority occurred in third decade (76 cases, 31.4%), with only few cases occurring beyond fifth decade (13 cases, 5.3%). Ganglioglioma/gangliocytoma (94 cases, 38.52%) was the most frequent followed by central neurocytoma (86 cases, 35.24%), paraganglioma (32 cases, 13.52%), dysembryoplastic neuroepithelial tumors (DNET) (21 cases, 8.6%), desmoplastic infantile astrocytoma/desmoplastic infantile ganglioglioma (DIA/DIG) (6 cases, 2.45%), papillary glioneuronal tumor (PGNT) (3 cases, 1.22%) and rosette-forming glioneuronal tumor (RGNT) (1 case, 0.4%). Association with seizures was noted in 40.95% of cases. Glioneuronal tumors are an expanding group of tumors with varying spectra of morphologic patterns and biological behavior. An improved understanding has direct clinical implications for optimizing current treatments and developing novel therapeutic approaches. Although most glioneuronal tumors carry a favorable prognosis, other factors such as inaccessibility to surgical resection and rarely, malignant transformation, make it difficult to accurately predict the biological behavior based on histopathology alone. Reliable prognostic markers remain to be defined.
Asunto(s)
Neoplasias del Sistema Nervioso Central/patología , Neoplasias Neuroepiteliales/patología , Adolescente , Adulto , Anciano , Neoplasias del Sistema Nervioso Central/epidemiología , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Neoplasias Neuroepiteliales/epidemiología , Adulto JovenRESUMEN
The hippocampus demonstrates age-associated changes in functions, neuronal circuitry, and plasticity during various developmental stages. On the contrary, there is a significant knowledge gap on age-associated proteomic alterations in the hippocampus subfields. Using tandem mass tag-based high-resolution mass spectrometry and quantitative proteomics, we report here age-associated changes in the human hippocampus at the subregional level. We used formalin-fixed paraffin-embedded hippocampal tissue sections from a total of 12 healthy individuals, with 3 individuals from each of the 4 different age groups, specifically, 1-10, 21-30, 31-40, and 81-90 years. We found that lysosome and oxidative phosphorylation were the pathways enriched in the 81- to 90-year age group. On the contrray, nervous system development, synaptic plasticity and transmission, messenger RNA (mRNA) splicing, and electron transport chain (ETC) complex-I activity were the enriched biological processes observed in the younger age groups. In a hippocampus subfield context, our topline findings on age-associated proteome changes include altered expression of proteins associated with adult neurogenesis with age in the dentate gyrus and increased expression of immune response-associated proteins with age in certain cornu ammonis sectors of the hippocampus. Signal peptide analysis predicted hippocampal proteins with secretory potential. While these new findings warrant replication in larger study samples, the current data contribute to (1) our understanding of the molecular basis of proteomic changes across various age groups in hippocampus subfields in healthy individuals, and (2) the design and interpretation of future research on the age-associated neurodegenerative disorders.
Asunto(s)
Imagen por Resonancia Magnética , Proteómica , Adulto , Anciano de 80 o más Años , Niño , Preescolar , Hipocampo/fisiología , Humanos , Lactante , Imagen por Resonancia Magnética/métodos , Proteoma , Adulto JovenRESUMEN
Lafora disease (LD) is the inherited progressive myoclonus epilepsy caused by mutations in either EPM2A gene, encoding the protein phosphatase laforin or the NHLRC1 gene, encoding the ubiquitin ligase malin. Since malin is an ubiquitin ligase and its mutations cause LD, it is hypothesized that improper clearance of its substrates might lead to LD pathogenesis. Here, we demonstrate for the first time that neuronatin is a novel substrate of malin. Malin interacts with neuronatin and enhances its degradation through proteasome. Interestingly, neuronatin is an aggregate prone protein, forms aggresome upon inhibition of cellular proteasome function and malin recruited to those aggresomes. Neuronatin is found to stimulate the glycogen synthesis through the activation of glycogen synthase and malin prevents neuronatin-induced glycogen synthesis. Several LD-associated mutants of malin are ineffective in the degradation of neuronatin and suppression of neuronatin-induced glycogen synthesis. Finally, we demonstrate the increased levels of neuronatin in the skin biopsy sample of LD patients. Overall, our results indicate that malin negatively regulates neuronatin and its loss of function in LD results in increased accumulation of neuronatin, which might be implicated in the formation of Lafora body or other aspect of disease pathogenesis.
Asunto(s)
Proteínas Portadoras/farmacología , Glucógeno/biosíntesis , Enfermedad de Lafora/enzimología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Animales , Western Blotting , Proteínas Portadoras/genética , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Enfermedad de Lafora/genética , Enfermedad de Lafora/metabolismo , Células PC12 , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/patología , Transfección , Ubiquitina-Proteína Ligasas , Ubiquitinación/efectos de los fármacosRESUMEN
The human hypothalamus is central to the regulation of neuroendocrine and neurovegetative systems, as well as modulation of chronobiology and behavioral aspects in human health and disease. Surprisingly, a deep proteomic analysis of the normal human hypothalamic proteome has been missing for such an important organ so far. In this study, we delineated the human hypothalamus proteome using a high-resolution mass spectrometry approach which resulted in the identification of 5349 proteins, while a multiple post-translational modification (PTM) search identified 191 additional proteins, which were missed in the first search. A proteogenomic analysis resulted in the discovery of multiple novel protein-coding regions as we identified proteins from noncoding regions (pseudogenes) and proteins translated from short open reading frames that can be missed using the traditional pipeline of prediction of protein-coding genes as a part of genome annotation. We also identified several PTMs of hypothalamic proteins that may be required for normal hypothalamic functions. Moreover, we observed an enrichment of proteins pertaining to autophagy and adult neurogenesis in the proteome data. We believe that the hypothalamic proteome reported herein would help to decipher the molecular basis for the diverse range of physiological functions attributed to it, as well as its role in neurological and psychiatric diseases. Extensive proteomic profiling of the hypothalamic nuclei would further elaborate on the role and functional characterization of several hypothalamus-specific proteins and pathways to inform future research and clinical discoveries in biological psychiatry, neurology, and system biology.
Asunto(s)
Psiquiatría Biológica , Proteómica , Adulto , Humanos , Hipotálamo/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/genética , Proteoma/metabolismo , Biología de SistemasRESUMEN
Neuropathology of drug resistant epilepsy (DRE) has direct bearing on the clinical outcome. Classification of the most common pathologies, hippocampal sclerosis (HS) and focal cortical dysplasia (FCD) have undergone several revisions and studies on the surgical pathology of DRE employing the updated ILAE classification are scarce. Here, we report the neuropathological spectrum of 482 surgically treated cases of DRE from a single institute using the latest ILAE classifications along with clinicoradiologic correlation. Majority of the cases (324, 67.2%) had temporal lobe epilepsy (TLE), with 158 (32.8%) having extratemporal seizure focus. Among TLE, HS was most common (n = 208, 64.2%), followed by neoplasms (42, 13%), FCD (26, 8%) and dual pathology (23, 7%). Less frequent were vascular malformations (cavernoma-3, arteriovenous malformation-1), mild malformation of cortical development (mMCD, 3), gliotic lesions (5), cysticercosis (2), double pathology (2) and polymicrogyria (1). Among extratemporal epilepsies, FCD was most common (46, 29.1%), followed by neoplasms (29, 18.3%), gliotic lesions (27, 17.1%), Rasmussen encephalitis (18, 11.4%), hypothalamic hamartoma (12, 7.6%), malformations of cortical development (10, 6.3%) and vascular malformations (6, 3.8%). Less frequent were double pathology (2, cysticercosis + FCD type IIb, DNET + FCD type IIb), mMCD (2), cysticercosis (1) and dual pathology (1). No underlying pathology was detected in 12 cases (2.5%). Radiopathological concordance was noted in 83%. In 36 cases (7.5%), histopathology detected an unsuspected second pathology that included FCD type III (n = 16) dual pathology (n = 18) and double pathology (n = 2). Further, in four MRI negative cases, histopathology was required for a conclusive diagnosis.
Asunto(s)
Epilepsia Refractaria , Epilepsia Refractaria/diagnóstico por imagen , Epilepsia Refractaria/epidemiología , Epilepsia Refractaria/cirugía , Humanos , Imagen por Resonancia Magnética , Malformaciones del Desarrollo Cortical/complicaciones , Malformaciones del Desarrollo Cortical/diagnóstico por imagen , Malformaciones del Desarrollo Cortical/epidemiología , Estudios Retrospectivos , Centros de Atención TerciariaRESUMEN
Atypical teratoid rhabdoid tumor (AT/RT) is a highly malignant embryonal CNS tumor, generally unresponsive to any form of therapy, uniformly fatal within 1 year. We report 15 cases of AT/RT diagnosed at our center over a period of 5 years (2003-08). Tumors were located in different sites of the neuraxis, posterior fossa being the most common (n = 10) followed by cerebral lobes (n = 3). There was one each at the supra sellar and cervical spinal regions, respectively. Radiologically most of the tumors were heterodense and enhancing heterogeneously. The tumors exhibited diverse histological profile that included rhabdoid and PNET areas in all cases, mesenchymal and epithelial areas in 73.3% and 53.3% cases, respectively. Necrosis was evident in all cases and one showed calcification. Tumor cells displayed a polyphenotypic immunoprofile. All cases were consistently positive for vimentin and epithelial membrane antigen and were negative for desmin. Variable positivity was seen for other markers. The number of cases positive for these were: CK (53%), SMA (60%), synaptophysin (66%), NFP (33.3%) and GFAP (85%). CK staining was prominent in epithelial areas, while PNET cells labeled prominently with synaptophysin. There was lack of INI1 expression in all cases. Follow-up was available in 46.6% of cases which revealed a uniform poor prognosis.
Asunto(s)
Neoplasias del Sistema Nervioso Central/química , Neoplasias del Sistema Nervioso Central/patología , Tumor Rabdoide/química , Tumor Rabdoide/patología , Teratoma/química , Teratoma/patología , Neoplasias del Sistema Nervioso Central/inmunología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , India , Lactante , Masculino , Necrosis , Estudios Retrospectivos , Tumor Rabdoide/inmunología , Teratoma/inmunologíaRESUMEN
Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20 LD families from the Indian population. We identified 12 distinct mutations in 15 LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210 K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Lafora/genética , Mutación , Proteínas Tirosina Fosfatasas no Receptoras/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Eliminación de Gen , Humanos , India , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación Missense , Linaje , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/química , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Alineación de Secuencia , Ubiquitina-Proteína LigasasRESUMEN
PURPOSE: The objective of this study was to study the altered proteome in the frontal lobe of patients with CM. Unbiased analysis of differentially abundant proteins could lead to identification of host responses against Plasmodium falciparum infection, which will aid in better understanding of the molecular mechanism of pathophysiology in CM. EXPERIMENTAL DESIGN: TMT-based quantitative proteomic analysis using high-resolution mass spectrometry is employed. In brief, proteins are isolated from frontal lobe samples, which are collected at autopsy from three cases of CM and three control subjects. Equal amounts of protein from each case are digested using trypsin and labeled with different TMT reagents. The pooled sample is fractionated using strong cation exchange chromatography and analyzed on Orbitrap Fusion in triplicates. For accurate quantitation of peptides, the samples are analyzed in MS3 mode. The data is searched against a combined database of human and P. falciparum proteins using Sequest and Mascot search engines. RESULTS: A total of 4174 proteins are identified, of which, 107 are found to be differentially abundant in the test samples with significant p-value (<0.05). Proteins associated with biological processes such as innate immune response, complement system, coagulation, and platelet activation are found to be elevated in CM cases. In contrast, proteins associated with myelination, oxidative phosphorylation, regulation of reactive oxygen species, and sodium and calcium ions transport are found to be depleted in response to CM. In addition, three P. falciparum proteins exclusively in CM brain samples are also identified. CONCLUSIONS AND CLINICAL RELEVANCE: The study signifies neuronal assault due to axonal injury, altered sodium and calcium ion channels, deregulated inflammation and demyelination as a part of host response to CM. Enhanced oxidative stress, repressed oxidative phosphorylation, and demyelination of axons may contribute to the severity of the disease. Further validation of these results on a large cohort can provide leads in the development of neuroprotective therapies for CM.
Asunto(s)
Biomarcadores/análisis , Encéfalo/metabolismo , Interacciones Huésped-Parásitos , Malaria Cerebral/metabolismo , Malaria Falciparum/metabolismo , Plasmodium falciparum/inmunología , Proteoma/análisis , Encéfalo/inmunología , Encéfalo/parasitología , Estudios de Casos y Controles , Humanos , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Proteómica/métodosRESUMEN
Healthy neurons do not store glycogen while they do possess the machinery for the glycogen synthesis albeit at an inactive state. Neurons in the degenerating brain, however, are known to accumulate glycogen, although its significance was not well understood. Emerging reports present contrasting views on neuronal glycogen synthesis; a few reports demonstrate a neurotoxic effect of glycogen while a few others suggest glycogen to be neuroprotective. Thus, the specific role of glycogen and glycogen synthase in neuronal physiology is largely unexplored. Using cellular and animal models of Huntington's disease, we show here that the overexpression of cytotoxic mutant huntingtin protein induces glycogen synthesis in the neurons by activating glycogen synthase and the overexpressed glycogen synthase protected neurons from the cytotoxicity of the mutant huntingtin. Exposure of neuronal cells to proteasomal blockade and oxidative stress also activate glycogen synthase to induce glycogen synthesis and to protect against stress-induced neuronal death. We show that the glycogen synthase plays an essential and inductive role in the neuronal autophagic flux, and helps in clearing the cytotoxic huntingtin aggregate. We also show that the increased neuronal glycogen inhibits the aggregation of mutant huntingtin, and thus could directly contribute to its clearance. Finally, we demonstrate that excessive autophagy flux is the molecular basis of cell death caused by the activation of glycogen synthase in unstressed neurons. Taken together, our results thus provide a novel function for glycogen synthase in proteolytic processes and offer insight into the role of glycogen synthase and glycogen in both survival and death of the neurons.