RESUMO
Brain function is critically dependent on correct circuit assembly. Microglia are well-known for their important roles in immunological defense and neural plasticity, but whether they can also mediate experience-induced correction of miswired circuitry is unclear. Ten-m3 knockout (KO) mice display a pronounced and stereotyped visuotopic mismapping of ipsilateral retinal inputs in their visual thalamus, providing a useful model to probe circuit correction mechanisms. Environmental enrichment (EE) commenced around birth, but not later in life, can drive a partial correction of the most mismapped retinal inputs in Ten-m3 KO mice. Here, we assess whether enrichment unlocks the capacity for microglia to selectively engulf and remove miswired circuitry, and the timing of this effect. Expression of the microglial-associated lysosomal protein CD68 showed a clear enrichment-driven, spatially restricted change which had not commenced at postnatal day (P)18, was evident at P21, more robust at P25, and had ceased by P30. This was observed specifically at the corrective pruning site and was absent at a control site. An engulfment assay at the corrective pruning site in P25 mice showed EE-driven microglial-uptake of the mismapped axon terminals. This was temporally and spatially specific, as no enrichment-driven microglial engulfment was seen in P18 KO mice, nor the control locus. The timecourse of the EE-driven corrective pruning as determined anatomically, aligned with this pattern of microglia reactivity and engulfment. Collectively, these findings show experience can drive targeted microglial engulfment of miswired neural circuitry during a restricted postnatal window. This may have important therapeutic implications for neurodevelopmental conditions involving aberrant neural connectivity.
Assuntos
Animais Recém-Nascidos , Camundongos Knockout , Microglia , Animais , Microglia/metabolismo , Microglia/fisiologia , Camundongos Endogâmicos C57BL , Camundongos , Plasticidade Neuronal/fisiologia , Antígenos CD/metabolismo , Vias Visuais/fisiologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Retina/fisiologia , Retina/citologia , Retina/metabolismo , Meio Ambiente , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/deficiência , Molécula CD68RESUMO
Environmental enrichment (EE) is beneficial for brain development and function, but our understanding of its capacity to drive circuit repair, the underlying mechanisms, and how this might vary with age remains limited. Ten-m3 knock-out (KO) mice exhibit a dramatic and stereotyped mistargeting of ipsilateral retinal inputs to the thalamus, resulting in visual deficits. We have recently shown a previously unexpected capacity for EE during early postnatal life (from birth for six weeks) to drive the partial elimination of miswired axonal projections, along with a recovery of visually mediated behavior, but the timeline of this repair was unclear. Here, we reveal that with just 3.5 weeks of EE from birth, Ten-m3 KOs exhibit a partial behavioral rescue, accompanied by pruning of the most profoundly miswired retinogeniculate terminals. Analysis suggests that the pruning is underway at this time point, providing an ideal opportunity to probe potential mechanisms. With the shorter EE-period, we found a localized increase in microglial density and activation profile within the identified geniculate region where corrective pruning was observed. No comparable response to EE was found in age-matched wild-type (WT) mice. These findings identify microglia as a potential mechanistic link through which EE drives the elimination of miswired neural circuits during early postnatal development. Activity driven, atypical recruitment of microglia to prune aberrant connectivity and restore function may have important therapeutic implications for neurodevelopmental disorders such as autistic spectrum disorder.
Assuntos
Axônios , Microglia , Animais , Camundongos , Camundongos Knockout , Microglia/fisiologia , Plasticidade Neuronal , Retina/fisiologia , Camundongos Endogâmicos C57BLRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor expressed by macrophages and microglia in the central nervous system (CNS). TREM2 has attracted a lot of interest in the past decade for its critical role in modulating microglia functions under homeostatic conditions and in neurodegenerative diseases. Genetic variation in TREM2 is sufficient to cause Nasu-Hakola disease, a rare pre-senile dementia with bone cysts, and to increase risk for Alzheimer's disease, frontotemporal dementia, and other neurodegenerative disorders. Beyond the role played by TREM2 genetic variants in these diseases, TREM2 engagement is a key step in microglia activation in response to different types of tissue injury (e.g. ß-Amyloid deposition, demyelination, apoptotic cell death) leading to enhanced microglia metabolism, phagocytosis, proliferation and survival. TREM2 also exists as a soluble form (sTREM2), generated from receptor shedding or alternative splicing, which is detectable in plasma and cerebrospinal fluid (CSF). Genetic variation, physiological conditions and disease status impact CSF sTREM2 levels. Clinical and preclinical studies suggest that targeting and/or monitoring sTREM2 could have clinical and therapeutic implications. Despite the critical role of sTREM2 in neurologic disease, its function remains poorly understood. Here, we review the current literature on sTREM2 regarding its origin, genetic variation, and possible functions as a biomarker in neurological disorders and as a potential active player in CNS diseases and target for therapies.
Assuntos
Doença de Alzheimer , Demência Frontotemporal , Doença de Alzheimer/metabolismo , Biomarcadores/metabolismo , Demência Frontotemporal/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Some aged community dogs acquire a degenerative syndrome termed Canine Cognitive Dysfunction (CCD) that resembles human dementia because of Alzheimer's Disease (AD), with comparable cognitive and behavioral deficits. Dogs also have similar neuroanatomy, share our domestic environment and develop amyloid-ß plaques, making them likely a valuable ecological model of AD. However, prior investigations have demonstrated a lack of neurofibrillary tau pathology in aged dogs, an important hallmark of AD, though elevated phosphorylated tau (p-tau) at the Serine 396 (S396) epitope has been reported in CCD. Here using enhanced immunohistochemical methods, we investigated p-tau in six CCD brains and six controls using the AT8 antibody (later stage neurofibrillary pathology), and an antibody against S396 p-tau (earlier stage tau dysfunction). For the first time, we systematically assessed the Papez circuit and regions associated with Braak staging and found that all CCD dogs displayed elevated S396 p-tau labeling throughout the circuit. The limbic thalamus was particularly implicated, with a similar labeling pattern to that reported for AD neurofibrillary pathology, especially the anterior nuclei, while the hippocampus exhibited dysfunction confined to synaptic layers and efferent pathways. The cingulate and temporal lobes displayed significantly greater tauopathy than the frontal and occipital cortices, also reflective of early Braak staging patterns in AD. Immunofluorescence confirmed that S396 was accumulating within neuronal axons, somata and oligodendrocytes. We also observed AT8 labeling in one CCD brain, near the transentorhinal cortex in layer II neurons, one of the first regions to be affected in AD. Together, these data demonstrate a concordance in regional distribution of tauopathy between CCD and AD, most evident in the limbic thalamus, an important step in further validating CCD as a translational model for human AD and understanding early AD pathogenic mechanisms.
Assuntos
Encéfalo/patologia , Disfunção Cognitiva/patologia , Doenças do Cão/patologia , Tauopatias/patologia , Proteínas tau/metabolismo , Doença de Alzheimer , Animais , Disfunção Cognitiva/metabolismo , Doenças do Cão/metabolismo , Cães , Feminino , Masculino , Fosforilação , Tauopatias/metabolismoRESUMO
Alzheimer's disease (AD) is a late-onset disease that has proved difficult to model. Microglia are implicated in AD, but reports vary on precisely when and how in the sequence of pathological changes they become involved. Here, post-mortem human tissue from two differentially affected regions of the AD brain and from non-demented individuals with a high load of AD-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-ß (Aß) and hyperphosphorylated microtubule associated protein tau pathology. Immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (IBA1), Aß and tau, was performed in the primary motor cortex (PMC), a region relatively spared of AD pathological changes, and compared to the severely affected inferior temporal cortex (ITC) in the same cases. Unlike the ITC, the PMC in the AD cases was spared of any degenerative changes in cortical thickness and the density of Betz cells and total neurons. The clustering of activated microglia was greatest in the PMC of AD cases and high pathology controls compared to the ITC. This suggests microglial activation is most prominent in the early phases of AD pathophysiology. Nascent tau inclusions were found in neuritic plaques in the PMC but were more numerous in the ITC of the same case. This shows that tau positive neuritic plaques begin early in AD which is likely of pathogenic importance, however major tau deposition follows the accumulation of Aß and clustering of activated microglia. Importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of Aß and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention.
RESUMO
Cerebrovascular pathology is common in aging and Alzheimer's disease (AD). The microvasculature is particularly vulnerable, with capillary-level microhemorrhages coinciding with amyloid beta deposits in senile plaques. In the current analysis, we assessed the relationship between cerebral microvessels and the neuritic component of the plaque in cortical and hippocampal 50- to 200-µm sections from 11 AD, 3 Down syndrome, and 7 nondemented cases in neuritic disease stages 0-VI. We report that 77%-97% of neuritic plaques are perivascular, independently of disease stage or dementia diagnosis. Within neuritic plaques, dystrophic hyperphosphorylated tau-positive neurites appear as clusters of punctate, bulbous, and thread-like structures focused around capillaries and colocalize with iron deposits characteristic of microhemorrhage. Microvessels within the neuritic plaque are narrowed by 1.0 ± 1.0 µm-4.4 ± 2.0 µm, a difference of 16%-65% compared to blood vessel segments with diameters 7.9 ± 2.0-6.4 ± 0.8 µm (p < 0.01) outside the plaque domain. The reduced capacity of microvessels within plaques, frequently below patency, likely compromises normal microlocal cerebrovascular perfusion. These data link the neuritic and amyloid beta components of the plaque directly to microvascular degeneration. Strategies focused on cerebrovascular antecedents to neuritic dystrophy in AD have immediate potential for prevention, detection, and therapeutic intervention.
Assuntos
Doença de Alzheimer/patologia , Sistema Glinfático/patologia , Microvasos/patologia , Neuritos/patologia , Placa Amiloide/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Sistema Glinfático/química , Humanos , Imageamento Tridimensional/métodos , Masculino , Microvasos/química , Pessoa de Meia-Idade , Neuritos/química , Neurônios/química , Neurônios/patologia , Placa Amiloide/químicaRESUMO
Microglial associations with both the major Alzheimer's disease (AD) pathognomonic entities, ß-amyloid-positive plaques and tau-positive neurofibrillary tangles, have been noted in previous investigations of both human tissue and mouse models. However, the precise nature of their role in the pathogenesis of AD is debated; the major working hypothesis is that pro-inflammatory activities of activated microglia contribute to disease progression. In contrast, others have proposed that microglial dystrophy with a loss of physiological and neuroprotective activities promotes neurodegeneration. This immunohistochemical study sought to gain clarity in this area by quantifying the morphological subtypes of microglia in the mildly-affected primary visual cortex (PVC), the moderately affected superior frontal cortex (SFC) and the severely affected inferior temporal cortex (ITC) of 8 AD cases and 15 age and gender-matched, non-demented controls with ranging AD-type pathology. AD cases had increased ß-amyloid and tau levels compared to controls in all regions. Neuronal loss was observed in the SFC and ITC, and was associated with atrophy in the latter. A major feature of the ITC in AD was a decrease in ramified (healthy) microglia with image analysis confirming reductions in arborized area and skeletal complexity. Activated microglia were not associated with AD but were increased in non-demented controls with greater AD-type pathology. Microglial clusters were occasionally associated with ß-amyloid- and tau-positive plaques but represented less than 2% of the total microglial population. Dystrophic microglia were not associated with AD, but were inversely correlated with brain pH suggesting that agonal events were responsible for this morphological subtype. Overall these novel findings suggest that there is an early microglial reaction to AD-type pathology but a loss of healthy microglia is the prominent feature in severely affected regions of the AD brain.
Assuntos
Doença de Alzheimer/patologia , Microglia/patologia , Tauopatias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Progressão da Doença , Feminino , Lobo Frontal/patologia , Humanos , Masculino , Doenças do Sistema Nervoso/patologia , Emaranhados Neurofibrilares/metabolismo , Neuropatologia , Placa Amiloide/patologia , Lobo Temporal/patologia , Córtex Visual/patologia , Proteínas tau/metabolismoRESUMO
We describe a protocol for culturing neurons from transgenic zebrafish embryos to investigate the subcellular distribution and protein aggregation status of neurodegenerative disease-causing proteins. The utility of the protocol was demonstrated on cell cultures from zebrafish that transgenically express disease-causing variants of human fused in sarcoma (FUS) and ataxin-3 proteins, in order to study amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-3 (SCA3), respectively. A mixture of neuronal subtypes, including motor neurons, exhibited differentiation and neurite outgrowth in the cultures. As reported previously, mutant human FUS was found to be mislocalized from nuclei to the cytosol, mimicking the pathology seen in human ALS and the zebrafish FUS model. In contrast, neurons cultured from zebrafish expressing human ataxin-3 with disease-associated expanded polyQ repeats did not accumulate within nuclei in a manner often reported to occur in SCA3. Despite this, the subcellular localization of the human ataxin-3 protein seen in cell cultures was similar to that found in the SCA3 zebrafish themselves. The finding of similar protein localization and aggregation status in the neuronal cultures and corresponding transgenic zebrafish models confirms that this cell culture model is a useful tool for investigating the cell biology and proteinopathy signatures of mutant proteins for the study of neurodegenerative disease.
RESUMO
The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD by expressing human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing) within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared with ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-ataxin-3-84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Cotreatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through macro-autophagy plays an important role in this beneficial effect.SIGNIFICANCE STATEMENT We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor compound calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.
Assuntos
Ataxina-3/metabolismo , Autofagia/fisiologia , Calpaína/metabolismo , Modelos Animais de Doenças , Glicoproteínas/farmacologia , Doença de Machado-Joseph/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Ataxina-3/genética , Autofagia/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Calpaína/genética , Feminino , Glicoproteínas/uso terapêutico , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/prevenção & controle , Masculino , Proteínas Repressoras/genética , Peixe-ZebraRESUMO
Changes in microglia function are involved in Alzheimer's disease (AD) for which ageing is the major risk factor. We evaluated microglial cell process morphologies and their gray matter coverage (arborized area) during ageing and in the presence and absence of AD pathology in autopsied human neocortex. Microglial cell processes were reduced in length, showed less branching and reduced arborized area with aging (case range 52-98 years). This occurred during normal ageing and without microglia dystrophy or changes in cell density. There was a larger reduction in process length and arborized area in AD compared to aged-matched control microglia. In AD cases, on average, 49%-64% of microglia had discontinuous and/or punctate Iba1 labeled processes instead of continuous Iba1 distribution. Up to 16% of aged-matched control microglia displayed discontinuous or punctate features. There was no change in the density of microglial cell bodies in gray matter during ageing or AD. This demonstrates that human microglia show progressive cell process retraction without cell loss during ageing. Additional changes in microglia occur with AD including Iba1 protein puncta and discontinuity. We suggest that reduced microglial arborized area may be an aging-related correlate of AD in humans. These variations in microglial cells during ageing and in AD could reflect changes in neural-glial interactions which are emerging as key to mechanisms involved in ageing and neurodegenerative disease.
Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Microglia/citologia , Autopsia , Encéfalo/patologia , Encéfalo/fisiologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Microglia/patologia , Neocórtex/metabolismo , Proteínas tau/metabolismoRESUMO
Alzheimer's disease (AD) involves the propagation of filaments of tau protein throughout the cerebral cortex. Imaging tau filaments and oligomers in human brain at high resolution would help contribute insight into the mechanism and progression of tauopathic diseases. STED microscopy is a nano-scale imaging technique and we aimed to test the abilities of this method for resolving tau structures within human brain. Using autopsied 50µm AD brain sections, we demonstrate that STED microscopy can resolve immunolabelled tau filaments at 77nm resolution. Ribbon-like tau filaments imaged by STED appeared smooth along their axis with limited axial undulations. STED also resolved 70-80nm wide tau puncta. Of the fluorophores tested, STAR635p was optimal for STED imaging in this tissue. This was in part due to brain tissue autofluorescence within the lower wavelength ranges (488-590nm). Further, the stability and minimal photobleaching of STAR635p allowed STED z-stacks of neurons packed with tau filaments (neurofibrillary tangles) to be collated. There was no loss of x-y image resolution of individual tau filaments through the 20µm z-stack. This demonstrates that STED can contribute to nano-scale analysis and characterisation of pathologies within banked human autopsied brain tissue. Resolving tau structures at this level of resolution provides promising avenues for understanding mechanisms of pathology propagation in the different tauopathies as well as illuminating what contributes to disease heterogeneity.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Substância Cinzenta/patologia , Proteínas tau/química , Doença de Alzheimer/patologia , Substância Cinzenta/diagnóstico por imagem , Humanos , Imageamento Tridimensional , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/ultraestrutura , Imagem Óptica , Coloração e Rotulagem , Proteínas tau/ultraestruturaRESUMO
BACKGROUND: Imaging of human brain as well as cellular and animal models has highlighted a role for the actin cytoskeleton in the development of cell pathology in Alzheimer's disease (AD). Rods and aggregates of the actin-associated protein cofilin are abundant in grey matter of postmortem AD brain and rods are found inside neurites in animal and cell models of AD. OBJECTIVE: We sought further understanding of the significance of cofilin rods/aggregates to the disease process: Do rods/aggregates correlate with AD progression and the development of hallmark neurofibrillary tangles and neuropil threads? Are cofilin rods/aggregates found in the same neurites as hyperphosphorylated tau? METHODS: The specificity of rods/aggregates to AD compared with general aging and their spatial relationship to tau protein was examined in postmortem human hippocampus, inferior temporal cortex, and anterior cingulate cortex. RESULTS: The presence of cofilin rods/aggregates correlated with the extent of tau pathology independent of patient age. Densities of rods/aggregates were fourfold greater in AD compared with aged-matched control brains and rods/aggregates were significantly larger in AD brain. We did not find evidence for our hypothesis that intracellular cofilin rods are localized to tau-positive neuropil threads. Instead, data suggest the involvement of microglia in the clearance of cofilin rods/aggregates and/or in their synthesis in and around amyloid plaques and surrounding neuropil. CONCLUSION: Cofilin rods and aggregates signify events initiated early in the pathological cascade. Further definition of the mechanisms leading to their formation in the human brain will provide insights into the cellular causes of AD.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Western Blotting , Encéfalo/irrigação sanguínea , Feminino , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imageamento Tridimensional , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Microglia/metabolismo , Microglia/patologia , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas tau/metabolismoRESUMO
FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Mutação , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Células Cultivadas , Humanos , Neurônios Motores/citologia , Transporte Proteico/genéticaRESUMO
Elevated levels of amyloid-ß (Aß) peptides, the main component of amyloid plaques in Alzheimer's disease, are the result of excessive ß- and γ-cleavage of the amyloid precursor protein (APP) and/or impaired Aß clearance in the brain. It has been suggested that high concentrations of luteinizing hormone (LH) in women contribute to increased Aß generation after menopause, but the mechanism for this is incompletely understood. We investigated the effect of human chorionic gonadotropin (hCG), an LH receptor agonist, on APP ß-cleavage in the SH-SY5Y neuroblastoma cell line. Treatment of these cells with hCG-induced elevated ß-cleavage in a dose-dependent manner: administration of 30 mIU but not 10 mIU/ml of hCG significantly increased sAPPß levels in the cell medium 1.7-fold as measured by ELISA. These results support the notion that LH contributes to elevated Aß levels at least in part by increasing ß-cleavage of APP by ß-site APP cleaving enzyme.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Gonadotropina Coriônica/farmacologia , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neuroblastoma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismoRESUMO
Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Epitopos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Western Blotting , Células Cultivadas , Embrião de Galinha , Galinhas , Humanos , Técnicas In Vitro , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microtúbulos/ultraestrutura , Mitocôndrias/metabolismo , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/ultraestrutura , FosforilaçãoRESUMO
Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).
Assuntos
Amiloide/química , Amiloide/ultraestrutura , Demência/patologia , Microscopia Eletrônica de Transmissão e Varredura/métodos , Modelos Moleculares , Fenótipo , HumanosRESUMO
The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nanoscale to the microscale. Importantly, the measurements are made in buffer solutions, allowing biological samples to "stay alive" within a physiological-like environment while temporal changes in structure are measured-e.g., before and after addition of chemical reagents. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, e.g., electron microscopy (EM). This unit provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins, cells, and tissues. The physical principles of the technique and methodological aspects of its practical use and applications are also described.
Assuntos
Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Silicatos de Alumínio/química , Grafite/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Proteínas/química , Proteínas/ultraestrutura , Eletricidade Estática , Propriedades de SuperfícieRESUMO
In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Neuritos/metabolismo , Neurônios/patologia , Proteínas tau/metabolismo , Fatores de Despolimerização de Actina/genética , Trifosfato de Adenosina/farmacologia , Doença de Alzheimer/patologia , Motivos de Aminoácidos/fisiologia , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Encéfalo/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , Embrião de Galinha/citologia , Depsipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Ionóforos/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Oxidantes/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosforilação/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Serina/metabolismo , Tiazolidinas/farmacologia , Transfecção/métodos , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.