RESUMEN
The blood-brain barrier (BBB) is essential for cerebral homeostasis and controls the selective passage of molecules traveling in and out of the brain. Despite the crucial role of the BBB in a variety of brain diseases and its relevance for the development of drugs, there is little known about its molecular architecture. In particular, the composition of the basal lamina between the astrocytic end-feet and the endothelial cells is only partly known. Here, we present a proteomic analysis of the basal lamina of the human BBB. We combined laser capture microdissection with shotgun proteomics for selective enrichment and identification of specific proteins present in the cerebral microvasculature and arachnoidal vessels collected from normal human brain tissue specimens. Proteins found to be associated with the blood-brain barrier were validated by immunohistochemistry. Expression of membrane protein MLC1 was found in all brain barriers. Phosphoglucomutase-like protein 5 appeared to be variably present along the outer part of intracerebral vessels, and multidrug resistance protein 1 was identified in both intracerebral, as well as arachnoidal blood vessels. The results demonstrate the presence of so far unidentified proteins in the human BBB and illustrate topic differences in their expression. In conclusion, we showed that sample purification by microdissection followed by shotgun proteomics provides a list of proteins identified in the BBB. Subsequent immunohistochemistry detailed the respective expression sites of membrane protein MLC1 and phosphoglucomutase-related protein 5. The role of the identified proteins in the functioning of the BBB needs further investigations.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Células Endoteliales , Proteómica , Transporte Biológico , Humanos , Proteínas/metabolismoRESUMEN
The unfolded protein response (UPR) is a stress response activated upon disturbed homeostasis in the endoplasmic reticulum (ER). Previously, we reported that the activation of the UPR closely correlates with the presence of phosphorylated tau (p-tau) in Alzheimer's disease (AD). As well as increased presence of intracellular p-tau, AD brains are characterized by extracellular deposits of ß amyloid (Aß). Recent in vitro studies have shown that Aß can induce ER stress and activation of the UPR. The aim of the present study is to investigate UPR activation in sporadic tauopathies like progressive supranuclear palsy (PSP) and Pick's disease (PiD), and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) which carry mutations in the gene encoding for tau (MAPT). The presence of phosphorylated pancreatic ER kinase (pPERK) and phosphorylated inositol requiring enzyme 1α (pIRE1), which are indicative of an activated UPR, was assessed by immunohistochemistry in cases neuropathologically defined as frontotemporal lobar degeneration with tau pathology (FTLD-tau). Increased presence of UPR activation markers pPERK and pIRE1 was observed in neurons and glia in FTLD-tau cases, in contrast to FTLD subtypes negative for tau pathology or in non-neurological controls. pPERK and pIRE1 were also prominently present in relatively young carriers of MAPT mutation. A strong association between the presence of UPR activation markers and p-tau was observed in the hippocampus of FTLD-tau cases. Double immunohistochemical staining on FTLD-tau cases revealed that UPR activation is predominantly observed in neurons that show diffuse staining of p-tau. These data demonstrate that UPR activation is intimately connected with the accumulation and aggregation of p-tau, and occurs independently from Aß deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies.
Asunto(s)
Hipocampo/química , Tauopatías/metabolismo , Respuesta de Proteína Desplegada , Proteínas tau/análisis , Adulto , Anciano , Anciano de 80 o más Años , Autopsia , Biomarcadores/análisis , Estudios de Casos y Controles , Endorribonucleasas/análisis , Femenino , Hipocampo/patología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Tauopatías/genética , Regulación hacia Arriba , eIF-2 Quinasa/análisis , Proteínas tau/genéticaRESUMEN
BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by the accumulation and aggregation of misfolded proteins. Disturbed homeostasis in the endoplasmic reticulum leads to accumulation of misfolded proteins, which triggers a stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. OBJECTIVE: In this paper, we will briefly review the early involvement of the UPR in the pathology of AD and PD. METHODS: Expression of UPR activation markers was analyzed in human brain tissue using immunohistochemistry and Western blot analysis. RESULTS: Neuropathological studies demonstrate that UPR activation markers are increased in neurons in AD and PD. In AD, UPR activation markers are observed in neurons with diffuse staining of phosphorylated tau protein. In PD, increased immunoreactivity for UPR activation markers is detected in neuromelanin containing dopaminergic neurons of the substantia nigra, which colocalize with diffuse α-synuclein staining. CONCLUSION: UPR activation is closely associated with the first stages of accumulation and aggregation of the toxic proteins involved in AD and PD. Studies of postmortem brain tissue indicate that UPR activation is an early event in neurodegeneration.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/patología , Retículo Endoplásmico/metabolismo , Neuronas/ultraestructura , Enfermedad de Parkinson/patología , Respuesta de Proteína Desplegada/fisiología , Enfermedad de Alzheimer/fisiopatología , Animales , Humanos , Neuronas/patología , Enfermedad de Parkinson/fisiopatologíaRESUMEN
Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2alpha, and inositol-requiring enzyme 1alpha was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3beta in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/patología , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Persona de Mediana Edad , Neuronas/patología , Fosforilación , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Sequestosoma-1 , Ubiquitina/metabolismo , eIF-2 Quinasa/metabolismo , Proteínas tau/metabolismoRESUMEN
Knowledge about the molecular mechanisms driving Alzheimer's disease (AD) is still limited. To learn more about AD biology, we performed whole transcriptome sequencing on the hippocampus of 20 AD cases and 10 age- and sex-matched cognitively healthy controls. We observed 2716 differentially expressed genes, of which 48% replicated in a second data set of 84 AD cases and 33 controls. We used an integrative network-based approach for combining transcriptomic and protein-protein interaction data to find differentially expressed gene modules that may reflect key processes in AD biology. A total of 735 differentially expressed genes were clustered into 33 modules, of which 82% replicated in a second data set, highlighting the robustness of this approach. These 27 modules were enriched for signal transduction, transport, response to stimulus, and several organic and cellular metabolic pathways. Ten modules interacted with previously described AD genes. Our study indicates that analyzing RNA-expression data based on annotated gene modules is more robust than on individual genes. We provide a comprehensive overview of the biological processes involved in AD, and the detected differentially expressed gene modules may provide a molecular basis for future research into mechanisms underlying AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Perfilación de la Expresión Génica , Hipocampo , Mapas de Interacción de Proteínas , Transducción de Señal/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/etiología , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
OBJECTIVE: To identify novel CSF biomarkers in GRN-associated frontotemporal dementia (FTD) by proteomics using mass spectrometry (MS). METHODS: Unbiased MS was applied to CSF samples from 19 presymptomatic and 9 symptomatic GRN mutation carriers and 24 noncarriers. Protein abundances were compared between these groups. Proteins were then selected for validation if identified by ≥4 peptides and if fold change was ≤0.5 or ≥2.0. Validation and absolute quantification by parallel reaction monitoring (PRM), a high-resolution targeted MS method, was performed on an international cohort (n = 210) of presymptomatic and symptomatic GRN, C9orf72 and MAPT mutation carriers. RESULTS: Unbiased MS revealed 20 differentially abundant proteins between symptomatic mutation carriers and noncarriers and nine between symptomatic and presymptomatic carriers. Seven of these proteins fulfilled our criteria for validation. PRM analyses revealed that symptomatic GRN mutation carriers had significantly lower levels of neuronal pentraxin receptor (NPTXR), receptor-type tyrosine-protein phosphatase N2 (PTPRN2), neurosecretory protein VGF, chromogranin-A (CHGA), and V-set and transmembrane domain-containing protein 2B (VSTM2B) than presymptomatic carriers and noncarriers. Symptomatic C9orf72 mutation carriers had lower levels of NPTXR, PTPRN2, CHGA, and VSTM2B than noncarriers, while symptomatic MAPT mutation carriers had lower levels of NPTXR and CHGA than noncarriers. INTERPRETATION: We identified and validated five novel CSF biomarkers in GRN-associated FTD. Our results show that synaptic, secretory vesicle, and inflammatory proteins are dysregulated in the symptomatic stage and may provide new insights into the pathophysiology of genetic FTD. Further validation is needed to investigate their clinical applicability as diagnostic or monitoring biomarkers.
Asunto(s)
Biomarcadores/líquido cefalorraquídeo , Demencia Frontotemporal/líquido cefalorraquídeo , Demencia Frontotemporal/genética , Proteómica , Adulto , Anciano , Proteína C9orf72/genética , Estudios de Cohortes , Femenino , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Enfermedad de Pick/líquido cefalorraquídeo , Enfermedad de Pick/genética , Proteómica/métodosRESUMEN
Laser capture microdissection (LCM) is used to isolate minute amounts of tissue for subsequent proteomics analysis. An advantage of this technique is that a specific subset of cells or tissue structures can be isolated and enriched from surrounding material. Here, we describe a LCM-based method for the isolation of amyloid plaques from the Alzheimer's disease (AD) brain using fluorescence and nano liquid chromatography (nLC) mass spectrometry (MS) analysis. Using this approach, we detect ~600 proteins in ~250 AD amyloid plaques.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/patología , Colorantes Fluorescentes/metabolismo , Captura por Microdisección con Láser/métodos , Espectrometría de Masas/métodos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Métodos Analíticos de la Preparación de la Muestra , Encéfalo/metabolismo , Cromatografía Liquida , Humanos , Nanotecnología , Coloración y EtiquetadoRESUMEN
Increased levels of pregnancy zone protein (PZP) were found in the serum of persons who later developed Alzheimer's disease (AD) in comparison to controls who remained dementia free. We suggested that this increase is due to brain derived PZP entering the blood stream during the early phase of the disease. Here we investigate the possible involvement of PZP in human AD pathogenesis. We observed increased PZP immunoreactivity in AD postmortem brain cortex compared to non-demented controls. In the AD cortex, PZP immunoreactivity localized to microglial cells that interacted with senile plaques and was occasionally observed in neurons. Our data link the finding of elevated serum PZP levels with the characteristic AD pathology and identify PZP as a novel component in AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Placa Amiloide/metabolismo , Placa Amiloide/patología , Proteínas Gestacionales/metabolismo , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Anticuerpos/metabolismo , Receptor 1 de Quimiocinas CX3C , Cromatografía Liquida , Diagnóstico , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunoprecipitación , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Fosfopiruvato Hidratasa/metabolismo , Proteínas Gestacionales/genética , Proteínas Gestacionales/inmunología , ARN Mensajero/metabolismo , Receptores de Quimiocina/metabolismo , Espectrometría de Masas en TándemRESUMEN
The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Lisosomas/enzimología , Respuesta de Proteína Desplegada/fisiología , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Células Cultivadas , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , RatasRESUMEN
Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore proteostasis in the ER. Previously, we demonstrated that UPR activation is an early event in Alzheimer disease (AD) brain. In our recent work we investigated whether activation of the UPR is employed to enhance the capacity of the ubiquitin proteasome system or autophagy in neuronal cells. We showed that the levels, composition and activity of the proteasome are not regulated by the UPR. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.