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APOEε4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). Although APOEε4 is known to promote Aß pathology, recent data also support an effect of APOE polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across APOE genotypes in the frontal cortex of 10 advanced AD cases (n = 5 APOEε3/ε3 and n = 5 APOEε4/ε4), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-Aß 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases (n = 11 APOEε3/ε3 and n = 10 APOEε4/ε4). Our dataset includes 1130 and 1330 proteins enriched in IPPHF1 samples from APOEε3/ε3 and APOEε4/ε4 groups (fold change ≥ 1.50, IPPHF1 vs IPIgG ctrl). We identified 80 and 68 proteins as probable pTau interactors in APOEε3/ε3 and APOEε4/ε4 groups, respectively (SAINT score ≥ 0.80; false discovery rate (FDR) ≤ 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOEε3/ε3 vs APOEε4/ε4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOEε4/ε4 vs APOEε3/ε3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOEε4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOEε4/ε4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to Aß-affected brain regions in APOEε4 carriers, paving the way to the identification of new therapeutic targets.
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Doença de Alzheimer , Apolipoproteína E4 , Proteínas tau , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Genótipo , Fosforilação , Proteômica , Proteínas tau/metabolismo , Proteínas tau/genéticaRESUMO
BACKGROUND: Subjective cognitive decline (SCD), considered a preclinical dementia stage, is less understood in Hispanics, a high-risk group for dementia. We investigated SCD to mild cognitive impairment (MCI) progression risk, as well as baseline and longitudinal features of depressive symptoms, SCD complaints, and objective cognitive performance among Hispanics compared to non-Hispanic Whites (NHW). METHODS: Hispanic (n = 23) and NHW (n = 165) SCD participants were evaluated at baseline and 2-year follow-up. Evaluations assessed function, depressive symptoms, SCD, and objective cognitive performance. RESULTS: Hispanics were at increased risk of progression to MCI (OR: 6.10, 95% CI 1.09-34.20, P = .040). Hispanic participants endorsed more depressive symptoms at baseline (P = .048) that worsened more longitudinally (OR: 3.16, 95% CI 1.18-8.51, P = .023). Hispanic participants had increased SCD complaints on the Brief Cognitive Rating Scale (BCRS) (ß = .40 SE: .17, P = .023), and in specific BCRS domains: concentration (ß = .13, SE: .07, P = .047), past memory (ß = .13, SE: .06, P = .039) and functional abilities (ß = .10, SE: .05, P = .037). In objective cognitive performance, Hispanic ethnicity associated with decline in MMSE (ß = -.27, SE: .13, P = .039), MoCA (ß = -.80 SE: .34, P = .032), Trails A (ß = 2.75, SE: .89, P = .002), Trails B (ß = 9.18, SE: 2.71, P = .001) and Guild Paragraph Recall Delayed (ß = -.80 SE: .28, P = .005). Conclusions: Hispanic ethnicity associated with a significantly increased risk of 2-year progression of SCD to MCI compared to NHW. This increased risk associated with increased depressive symptoms, distinctive SCD features, and elevated amnestic and non-amnestic objective cognitive decline. This supports further research to refine the assessment of preclinical dementia in this high-risk group.
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AIMS: Hippocampal findings are implicated in the pathogenesis of sudden unexplained death in childhood (SUDC), although some studies have identified similar findings in sudden explained death in childhood (SEDC) cases. We blindly reviewed hippocampal histology in SUDC and SEDC controls. METHODS: Hippocampal haematoxylin and eosin (H&E) slides (n = 67; 36 SUDC, 31 controls) from clinical and forensic collaborators were evaluated by nine blinded reviewers: three board-certified forensic pathologists, three neuropathologists and three dual-certified neuropathologists/forensic pathologists. RESULTS: Among nine reviewers, about 50% of hippocampal sections were rated as abnormal (52.5% SUDC, 53.0% controls), with no difference by cause of death (COD) (p = 0.16) or febrile seizure history (p = 0.90). There was little agreement among nine reviewers on whether a slide was within normal range (Fleiss' κ = 0.014, p = 0.47). Within reviewer groups, there were no findings more frequent in SUDC compared with controls, with variability in pyramidal neuron and dentate gyrus findings. Across reviewer groups, there was concordance for bilamination and granule cell loss. Neither SUDC (51.2%) nor control (55.9%) slides were considered contributory to determining COD (p = 0.41). CONCLUSIONS: The lack of an association of hippocampal findings in SUDC and controls, as well as inconsistency of observations by multiple blinded reviewers, indicates discrepancy with previous studies and an inability to reliably identify hippocampal maldevelopment associated with sudden death (HMASD). These findings underscore a need for larger studies to standardise evaluation of hippocampal findings, identifying the range of normal variation and changes unrelated to SUDC or febrile seizures. Molecular studies may help identify novel immunohistological markers that inform on COD.
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Neuropatologia , Convulsões Febris , Encéfalo/patologia , Criança , Morte Súbita/patologia , Hipocampo/patologia , Humanos , Convulsões Febris/complicações , Convulsões Febris/patologiaRESUMO
Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10-15, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10-21, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10-6, z = 2.65) and CA1-3 (p = 4.7 × 10-6, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10-5) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10-3) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10-5) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10-3). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.
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Proteômica , Convulsões Febris , Autopsia , Criança , Morte Súbita/etiologia , Morte Súbita/patologia , Hipocampo/patologia , Humanos , Convulsões Febris/complicações , Convulsões Febris/patologiaRESUMO
OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.
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Dengue/complicações , Dengue/patologia , Encefalite Viral/etiologia , Encefalite Viral/patologia , Doença Crônica , Demência , Vírus da Dengue , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Rapidly progressive Alzheimer's disease (rpAD) is a particularly aggressive form of Alzheimer's disease, with a median survival time of 7-10 months after diagnosis. Why these patients have such a rapid progression of Alzheimer's disease is currently unknown. To further understand pathological differences between rpAD and typical sporadic Alzheimer's disease (sAD) we used localized proteomics to analyze the protein differences in amyloid plaques in rpAD and sAD. Label-free quantitative LC-MS/MS was performed on amyloid plaques microdissected from rpAD and sAD patients (n = 22 for each patient group) and protein expression differences were quantified. On average, 913 ± 30 (mean ± SEM) proteins were quantified in plaques from each patient and 279 of these proteins were consistently found in plaques from every patient. We found significant differences in protein composition between rpAD and sAD plaques. We found that rpAD plaques contained significantly higher levels of neuronal proteins (p = 0.0017) and significantly lower levels of astrocytic proteins (p = 1.08 × 10-6). Unexpectedly, cumulative protein differences in rpAD plaques did not suggest accelerated typical sAD. Plaques from patients with rpAD were particularly abundant in synaptic proteins, especially those involved in synaptic vesicle release, highlighting the potential importance of synaptic dysfunction in the accelerated development of plaque pathology in rpAD. Combined, our data provide new direct evidence that amyloid plaques do not all have the same protein composition and that the proteomic differences in plaques could provide important insight into the factors that contribute to plaque development. The cumulative protein differences in rpAD plaques suggest rpAD may be a novel subtype of Alzheimer's disease.
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Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Placa Amiloide/metabolismo , Proteoma , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/patologia , Cromatografia Líquida , Estudos de Coortes , Progressão da Doença , Feminino , Imunofluorescência , Humanos , Masculino , Microdissecção , Microscopia Confocal , Pessoa de Meia-Idade , Neuritos/metabolismo , Neuritos/patologia , Placa Amiloide/patologia , Proteômica , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Isocitrate dehydrogenase (IDH) mutant astrocytoma grading, until recently, has been entirely based on morphology. The 5th edition of the Central Nervous System World Health Organization (WHO) introduces CDKN2A/B homozygous deletion as a biomarker of grade 4. We sought to investigate the prognostic impact of DNA methylation-derived molecular biomarkers for IDH mutant astrocytoma. METHODS: We analyzed 98 IDH mutant astrocytomas diagnosed at NYU Langone Health between 2014 and 2022. We reviewed DNA methylation subclass, CDKN2A/B homozygous deletion, and ploidy and correlated molecular biomarkers with histological grade, progression free (PFS), and overall (OS) survival. Findings were confirmed using 2 independent validation cohorts. RESULTS: There was no significant difference in OS or PFS when stratified by histologic WHO grade alone, copy number complexity, or extent of resection. OS was significantly different when patients were stratified either by CDKN2A/B homozygous deletion or by DNA methylation subclass (P valueâ =â .0286 and .0016, respectively). None of the molecular biomarkers were associated with significantly better PFS, although DNA methylation classification showed a trend (P valueâ =â .0534). CONCLUSIONS: The current WHO recognized grading criteria for IDH mutant astrocytomas show limited prognostic value. Stratification based on DNA methylation shows superior prognostic value for OS.
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Astrocitoma , Biomarcadores Tumorais , Neoplasias Encefálicas , Inibidor p16 de Quinase Dependente de Ciclina , Metilação de DNA , Isocitrato Desidrogenase , Mutação , Humanos , Astrocitoma/genética , Astrocitoma/patologia , Astrocitoma/mortalidade , Isocitrato Desidrogenase/genética , Masculino , Prognóstico , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/mortalidade , Adulto , Inibidor de Quinase Dependente de Ciclina p15/genética , Idoso , Taxa de Sobrevida , Seguimentos , Adulto Jovem , Homozigoto , Deleção de GenesRESUMO
IMPORTANCE: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. METHODS: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes. DISCUSSION: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC.
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COVID-19 , Adulto , Humanos , SARS-CoV-2 , Estudos Transversais , Síndrome de COVID-19 Pós-Aguda , Progressão da Doença , Fatores de RiscoRESUMO
Introduction: Alzheimer's disease (AD) and epilepsy are reciprocally related. Among sporadic AD patients, clinical seizures occur in 10-22% and subclinical epileptiform abnormalities occur in 22-54%. Cognitive deficits, especially short-term memory impairments, occur in most epilepsy patients. Common neurophysiological and molecular mechanisms occur in AD and epilepsy. The choroid plexus undergoes pathological changes in aging, AD, and epilepsy, including decreased CSF turnover, amyloid beta (Aß), and tau accumulation due to impaired clearance and disrupted CSF amino acid homeostasis. This pathology may contribute to synaptic dysfunction in AD and epilepsy. Methods: We evaluated control (n = 8), severe AD (n = 8; A3, B3, C3 neuropathology), and epilepsy autopsy cases (n = 12) using laser capture microdissection (LCM) followed by label-free quantitative mass spectrometry on the choroid plexus adjacent to the hippocampus at the lateral geniculate nucleus level. Results: Proteomics identified 2,459 proteins in the choroid plexus. At a 5% false discovery rate (FDR), 616 proteins were differentially expressed in AD vs. control, 1 protein in epilepsy vs. control, and 438 proteins in AD vs. epilepsy. There was more variability in the epilepsy group across syndromes. The top 20 signaling pathways associated with differentially expressed proteins in AD vs. control included cell metabolism pathways; activated fatty acid beta-oxidation (p = 2.00 x 10-7, z = 3.00), and inhibited glycolysis (p = 1.00 x 10-12, z = -3.46). For AD vs. epilepsy, the altered pathways included cell metabolism pathways, activated complement system (p = 5.62 x 10-5, z = 2.00), and pathogen-induced cytokine storm (p = 2.19 x 10-2, z = 3.61). Of the 617 altered proteins in AD and epilepsy vs. controls, 497 (81%) were positively correlated (p < 0.0001, R2 = 0.27). Discussion: We found altered signaling pathways in the choroid plexus of severe AD cases and many correlated changes in the protein expression of cell metabolism pathways in AD and epilepsy cases. The shared molecular mechanisms should be investigated further to distinguish primary pathogenic changes from the secondary ones. These mechanisms could inform novel therapeutic strategies to prevent disease progression or restore normal function. A focus on dual-diagnosed AD/epilepsy cases, specific epilepsy syndromes, such as temporal lobe epilepsy, and changes across different severity levels in AD and epilepsy would add to our understanding.
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Background: Central nervous system (CNS) cancer is the 10th leading cause of cancer-associated deaths for adults, but the leading cause in pediatric patients and young adults. The variety and complexity of histologic subtypes can lead to diagnostic errors. DNA methylation is an epigenetic modification that provides a tumor type-specific signature that can be used for diagnosis. Methods: We performed a prospective study using DNA methylation analysis as a primary diagnostic method for 1921 brain tumors. All tumors received a pathology diagnosis and profiling by whole genome DNA methylation, followed by next-generation DNA and RNA sequencing. Results were stratified by concordance between DNA methylation and histopathology, establishing diagnostic utility. Results: Of the 1602 cases with a World Health Organization histologic diagnosis, DNA methylation identified a diagnostic mismatch in 225 cases (14%), 78 cases (5%) did not classify with any class, and in an additional 110 (7%) cases DNA methylation confirmed the diagnosis and provided prognostic information. Of 319 cases carrying 195 different descriptive histologic diagnoses, DNA methylation provided a definitive diagnosis in 273 (86%) cases, separated them into 55 methylation classes, and changed the grading in 58 (18%) cases. Conclusions: DNA methylation analysis is a robust method to diagnose primary CNS tumors, improving diagnostic accuracy, decreasing diagnostic errors and inconclusive diagnoses, and providing prognostic subclassification. This study provides a framework for inclusion of DNA methylation profiling as a primary molecular diagnostic test into professional guidelines for CNS tumors. The benefits include increased diagnostic accuracy, improved patient management, and refinements in clinical trial design.
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Resolving glial contributions to Alzheimer's disease (AD) is necessary because changes in neuronal function, such as reduced synaptic density, altered electrophysiological properties, and degeneration, are not entirely cell autonomous. To improve understanding of transcriptomic heterogeneity in glia during AD, we used single-nuclei RNA sequencing (snRNA-seq) to characterize astrocytes and oligodendrocytes from apolipoprotein (APOE) Æ2/3 human AD and age- and genotype-matched non-symptomatic (NS) brains. We enriched astrocytes before sequencing and characterized pathology from the same location as the sequenced material. We characterized baseline heterogeneity in both astrocytes and oligodendrocytes and identified global and subtype-specific transcriptomic changes between AD and NS astrocytes and oligodendrocytes. We also took advantage of recent human and mouse spatial transcriptomics resources to localize heterogeneous astrocyte subtypes to specific regions in the healthy and inflamed brain. Finally, we integrated our data with published AD snRNA-seq datasets, highlighting the power of combining datasets to resolve previously unidentifiable astrocyte subpopulations.
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Doença de Alzheimer , Astrócitos , Doença de Alzheimer/patologia , Animais , Astrócitos/patologia , Humanos , Camundongos , Neuroglia/patologia , Oligodendroglia/patologia , RNA Nuclear Pequeno , TranscriptomaRESUMO
Amyloid plaques contain many proteins in addition to beta amyloid (Aß). Previous studies examining plaque-associated proteins have shown these additional proteins are important; they provide insight into the factors that drive amyloid plaque development and are potential biomarkers or therapeutic targets for Alzheimer's disease (AD). The aim of this study was to comprehensively identify proteins that are enriched in amyloid plaques using unbiased proteomics in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. We focused our study on early onset AD as the drivers of the more aggressive pathology development in these cases is unknown and it is unclear whether amyloid-plaque enriched proteins differ between subtypes of early onset AD. Amyloid plaques and neighbouring non-plaque tissue were microdissected from human brain sections using laser capture microdissection and label-free LC-MS was used to quantify the proteins present. 48 proteins were consistently enriched in amyloid plaques in EOAD and DS. Many of these proteins were more significantly enriched in amyloid plaques than Aß. The most enriched proteins in amyloid plaques in both EOAD and DS were: COL25A1, SMOC1, MDK, NTN1, OLFML3 and HTRA1. Endosomal/lysosomal proteins were particularly highly enriched in amyloid plaques. Fluorescent immunohistochemistry was used to validate the enrichment of four proteins in amyloid plaques (moesin, ezrin, ARL8B and SMOC1) and to compare the amount of total Aß, Aß40, Aß42, phosphorylated Aß, pyroglutamate Aß species and oligomeric species in EOAD and DS. These studies showed that phosphorylated Aß, pyroglutamate Aß species and SMOC1 were significantly higher in DS plaques, while oligomers were significantly higher in EOAD. Overall, we observed that amyloid plaques in EOAD and DS largely contained the same proteins, however the amount of enrichment of some proteins was different in EOAD and DS. Our study highlights the significant enrichment of many proteins in amyloid plaques, many of which may be potential therapeutic targets and/or biomarkers for AD.
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Doença de Alzheimer , Síndrome de Down , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Síndrome de Down/patologia , Glicoproteínas , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Placa Amiloide/patologia , Proteoma/metabolismo , Ácido PirrolidonocarboxílicoRESUMO
Brainstem nuclei dysfunction is implicated in sudden unexpected death in epilepsy. In animal models, deficient serotonergic activity is associated with seizure-induced respiratory arrest. In humans, glia are decreased in the ventrolateral medullary pre-Botzinger complex that modulate respiratory rhythm, as well as in the medial medullary raphe that modulate respiration and arousal. Finally, sudden unexpected death in epilepsy cases have decreased midbrain volume. To understand the potential role of brainstem nuclei in sudden unexpected death in epilepsy, we evaluated molecular signalling pathways using localized proteomics in microdissected midbrain dorsal raphe and medial medullary raphe serotonergic nuclei, as well as the ventrolateral medulla in brain tissue from epilepsy patients who died of sudden unexpected death in epilepsy and other causes in diverse epilepsy syndromes and non-epilepsy control cases (n = 15-16 cases per group/region). Compared with the dorsal raphe of non-epilepsy controls, we identified 89 proteins in non-sudden unexpected death in epilepsy and 219 proteins in sudden unexpected death in epilepsy that were differentially expressed. These proteins were associated with inhibition of EIF2 signalling (P-value of overlap = 1.29 × 10-8, z = -2.00) in non-sudden unexpected death in epilepsy. In sudden unexpected death in epilepsy, there were 10 activated pathways (top pathway: gluconeogenesis I, P-value of overlap = 3.02 × 10-6, z = 2.24) and 1 inhibited pathway (fatty acid beta-oxidation, P-value of overlap = 2.69 × 10-4, z = -2.00). Comparing sudden unexpected death in epilepsy and non-sudden unexpected death in epilepsy, 10 proteins were differentially expressed, but there were no associated signalling pathways. In both medullary regions, few proteins showed significant differences in pairwise comparisons. We identified altered proteins in the raphe and ventrolateral medulla of epilepsy patients, including some differentially expressed in sudden unexpected death in epilepsy cases. Altered signalling pathways in the dorsal raphe of sudden unexpected death in epilepsy indicate a shift in cellular energy production and activation of G-protein signalling, inflammatory response, stress response and neuronal migration/outgrowth. Future studies should assess the brain proteome in relation to additional clinical variables (e.g. recent tonic-clonic seizures) and in more of the reciprocally connected cortical and subcortical regions to better understand the pathophysiology of epilepsy and sudden unexpected death in epilepsy.
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Sudden unexpected death in epilepsy is the leading category of epilepsy-related death and the underlying mechanisms are incompletely understood. Risk factors can include a recent history and high frequency of generalized tonic-clonic seizures, which can depress brain activity postictally, impairing respiration, arousal and protective reflexes. Neuropathological findings in sudden unexpected death in epilepsy cases parallel those in other epilepsy patients, with no implication of novel structures or mechanisms in seizure-related deaths. Few large studies have comprehensively reviewed whole brain examination of such patients. We evaluated 92 North American Sudden unexpected death in epilepsy Registry cases with whole brain neuropathological examination by board-certified neuropathologists blinded to the adjudicated cause of death, with an average of 16 brain regions examined per case. The 92 cases included 61 sudden unexpected death in epilepsy (40 definite, 9 definite plus, 6 probable, 6 possible) and 31 people with epilepsy controls who died from other causes. The mean age at death was 34.4 years and 65.2% (60/92) were male. The average age of death was younger for sudden unexpected death in epilepsy cases than for epilepsy controls (30.0 versus 39.6 years; P = 0.006), and there was no difference in sex distribution respectively (67.3% male versus 64.5%, P = 0.8). Among sudden unexpected death in epilepsy cases, earlier age of epilepsy onset positively correlated with a younger age at death (P = 0.0005) and negatively correlated with epilepsy duration (P = 0.001). Neuropathological findings were identified in 83.7% of the cases in our cohort. The most common findings were dentate gyrus dysgenesis (sudden unexpected death in epilepsy 50.9%, epilepsy controls 54.8%) and focal cortical dysplasia (FCD) (sudden unexpected death in epilepsy 41.8%, epilepsy controls 29.0%). The neuropathological findings in sudden unexpected death in epilepsy paralleled those in epilepsy controls, including the frequency of total neuropathological findings as well as the specific findings in the dentate gyrus, findings pertaining to neurodevelopment (e.g. FCD, heterotopias) and findings in the brainstem (e.g. medullary arcuate or olivary dysgenesis). Thus, like prior studies, we found no neuropathological findings that were more common in sudden unexpected death in epilepsy cases. Future neuropathological studies evaluating larger sudden unexpected death in epilepsy and control cohorts would benefit from inclusion of different epilepsy syndromes with detailed phenotypic information, consensus among pathologists particularly for more subjective findings where observations can be inconsistent, and molecular approaches to identify markers of sudden unexpected death in epilepsy risk or pathogenesis.
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Epilepsy is a common neurological disorder affecting over 70 million people worldwide, with a high rate of pharmaco-resistance, diverse comorbidities including progressive cognitive and behavioural disorders, and increased mortality from direct (e.g. sudden unexpected death in epilepsy, accidents, drowning) or indirect effects of seizures and therapies. Extensive research with animal models and human studies provides limited insights into the mechanisms underlying seizures and epileptogenesis, and these have not translated into significant reductions in pharmaco-resistance, morbidities or mortality. To help define changes in molecular signalling networks associated with seizures in epilepsy with a broad range of aetiologies, we examined the proteome of brain samples from epilepsy and control cases. Label-free quantitative mass spectrometry was performed on the hippocampal cornu ammonis 1-3 region (CA1-3), frontal cortex and dentate gyrus microdissected from epilepsy and control cases (n = 14/group). Epilepsy cases had significant differences in the expression of 777 proteins in the hippocampal CA1 - 3 region, 296 proteins in the frontal cortex and 49 proteins in the dentate gyrus in comparison to control cases. Network analysis showed that proteins involved in protein synthesis, mitochondrial function, G-protein signalling and synaptic plasticity were particularly altered in epilepsy. While protein differences were most pronounced in the hippocampus, similar changes were observed in other brain regions indicating broad proteomic abnormalities in epilepsy. Among the most significantly altered proteins, G-protein subunit beta 1 (GNB1) was one of the most significantly decreased proteins in epilepsy in all regions studied, highlighting the importance of G-protein subunit signalling and G-protein-coupled receptors in epilepsy. Our results provide insights into common molecular mechanisms underlying epilepsy across various aetiologies, which may allow for novel targeted therapeutic strategies.
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OBJECTIVE: To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to control patients with epilepsy. METHODS: For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected postmortem brain tissue of 12 patients with SUDEP and 14 with non-SUDEP epilepsy. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from patients with mesial temporal lobe epilepsy: 6 low-risk and 8 high-risk SUDEP as determined by a short (<50 seconds) or prolonged (≥50 seconds) postictal generalized EEG suppression (PGES) that may indicate severely depressed brain activity impairing respiration, arousal, and protective reflexes. RESULTS: In autopsy hippocampus and cortex, we observed no proteomic differences between patients with SUDEP and those with non-SUDEP epilepsy, contrasting with our previously reported robust differences between epilepsy and controls without epilepsy. Transcriptomics in hippocampus and cortex from patients with surgical epilepsy segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 long noncoding RNAs, 3 pending) in hippocampus. CONCLUSION: The SUDEP proteome and high-risk SUDEP transcriptome were similar to those in other patients with epilepsy in hippocampus and cortex, consistent with diverse epilepsy syndromes and comorbid conditions associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions, may identify molecular mechanisms of SUDEP.
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Córtex Cerebral/fisiopatologia , Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Morte Súbita Inesperada na Epilepsia , Adulto , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Transdução de Sinais/fisiologiaRESUMO
Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.
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Encéfalo/metabolismo , Variações do Número de Cópias de DNA , Metilação de DNA , Epilepsia Resistente a Medicamentos/genética , Receptores ErbB/genética , Adolescente , Adulto , Criança , Epilepsia Resistente a Medicamentos/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
OBJECTIVES: Clinically relevant neuroanatomy is challenging to teach, learn and remember since many functionally important structures are visualized best using histology stains from serial 2D planar sections of the brain. In clinical patients, the locations of specific structures then must be inferred from spatial position and surface anatomy. A 3D MRI dataset of neuroanatomy has several advantages including simultaneous multi-planar visualization in the same brain, direct end-user manipulation of the data and image contrast identical to clinical MRI. We created 3D MRI datasets of the postmortem brain with high spatial and contrast resolution for simultaneous multi-planar visualization of complex neuroanatomy. MATERIALS AND METHODS: Whole human brains (N = 6) were immersion-fixed in 4% formaldehyde for 4 weeks, then washed continuously in water for 48 h. The brains were imaged on a clinical 3-T MRI scanner with a 64-channel head and neck coil using a 3D T2-weighted sequence with 400-micron isotropic resolution (voxel = 0.064 mm3; time = 7 h). Besides resolution, this sequence has multiple adjustments to improve contrast compared to a clinical protocol, including 93% reduced turbo factor and 77% reduced effective echo time. RESULTS: This MRI microscopy protocol provided excellent contrast resolution of small nuclei and internal myelinated pathways within the basal ganglia, thalamus, brainstem, and cerebellum. Contrast was sufficient to visualize the presence and variation of horizontal layers in the cerebral cortex. 3D isotropic resolution datasets facilitated simultaneous multi-planar visualization and efficient production of specific tailored oblique image orientations to improve understanding of complex neuroanatomy. CONCLUSION: We created an unlabeled high-resolution digital 3D MRI dataset of neuroanatomy as an online resource for readers to download, manipulate, annotate and use for clinical practice, research, and teaching that is complementary to traditional histology-based atlases. Digital MRI contrast is quantifiable, reproducible across brains and could help validate novel MRI strategies for in vivo structure visualization.
RESUMO
Sudden unexplained death in childhood (SUDC) affects children >1-year-old whose cause of death remains unexplained following comprehensive case investigation and is often associated with hippocampal abnormalities. We prospectively performed systematic neuropathologic investigation in 20 SUDC cases, including (i) autopsy data and comprehensive ancillary testing, including molecular studies, (ii) ex vivo 3T MRI and extensive histologic brain samples, and (iii) blinded neuropathology review by 2 board-certified neuropathologists. There were 12 girls and 8 boys; median age at death was 33.3 months. Twelve had a history of febrile seizures, 85% died during apparent sleep and 80% in prone position. Molecular testing possibly explained 3 deaths and identified genetic mutations in TNNI3, RYR2, and multiple chromosomal aberrations. Hippocampal abnormalities most often affected the dentate gyrus (altered thickness, irregular configuration, and focal lack of granule cells), and had highest concordance between reviewers. Findings were identified with similar frequencies in cases with and without molecular findings. Number of seizures did not correlate with hippocampal findings. Hippocampal alterations were the most common finding on histological review but were also found in possibly explained deaths. The significance and specificity of hippocampal findings is unclear as they may result from seizures, contribute to seizure pathogenesis, or be an unrelated phenomenon.