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1.
J Neuroinflammation ; 15(1): 256, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189875

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aß) known as plaques and intracellular tau tangles. Coincident with the formation of Aß plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aß deposition. METHODS: In this study, we utilized two animal models of AD to examine and characterize the AD-associated pathology: the Tg2576 Swedish APP (KM670/671NL) and TgCRND8 Swedish plus Indiana APP (KM670/671NL + V717F) lines. We used laser capture microscopy (LCM) to isolate samples surrounding Thio-S positive plaques from distal non-plaque tissue. These samples were then analyzed using RNA sequencing. RESULTS: We determined age-associated transcriptomic differences between two similar yet distinct APP transgenic mouse models, known to differ in proportional amyloidogenic species and plaque deposition rates. In Tg2576, human AD gene signatures were not observed despite profiling mice out to 15 months of age. TgCRND8 mice however showed progressive and robust induction of lysomal, neuroimmune, and ITIM/ITAM-associated gene signatures overlapping with prior human AD brain transcriptomic studies. Notably, RNAseq analyses highlighted the vast majority of transcriptional changes observed in aging TgCRND8 cortical brain homogenates were in fact specifically enriched within the plaque niche samples. Data uncovered plaque-associated enrichment of microglia-related genes such as ITIM/ITAM-associated genes and pathway markers of phagocytosis. CONCLUSION: This work may help guide improved translational value of APP mouse models of AD, particularly for strategies aimed at targeting neuroimmune and neurodegenerative pathways, by demonstrating that TgCRND8 more closely recapitulates specific human AD-associated transcriptional responses.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/genética , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/patologia , Correlação de Dados , Modelos Animais de Doenças , Humanos , Microdissecção e Captura a Laser , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Placa Amiloide/patologia , RNA Mensageiro/metabolismo , Transcriptoma
2.
Mol Neurodegener ; 12(1): 39, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28521765

RESUMO

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.


Assuntos
Tauopatias/metabolismo , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Piranos/farmacologia , Tiazóis/farmacologia
3.
J Comp Neurol ; 505(4): 352-62, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17912741

RESUMO

Mice with knock-in of two mutations that affect beta amyloid processing and levels (2xKI) exhibit impaired spatial memory by 9-12 months of age, together with synaptic plasticity dysfunction in the hippocampus. The goal of this study was to identify changes in the molecular and structural characteristics of synapses that precede and thus could exert constraints upon cellular mechanisms underlying synaptic plasticity. Drebrin A is one protein reported to modulate spine sizes and trafficking of proteins to and from excitatory synapses. Thus, we examined levels of drebrin A within postsynaptic spines in the hippocampus and entorhinal cortex. Our electron microscopic immunocytochemical analyses reveal that, by 6 months, the proportion of hippocampal spines containing drebrin A is reduced and this change is accompanied by an increase in the mean size of spines and decreased density of spines. In the entorhinal cortex of 2xKI brains, we detected no decrement in the proportion of spines labeled for drebrin A and no significant change in spine density at 6 months, but rather a highly significant reduction in the level of drebrin A immunoreactivity within each spine. These changes are unlike those observed for the somatosensory cortex of 2xKI mice, in which synapse density and drebrin A immunoreactivity levels remain unchanged at 6 months and older. These results indicate that brains of 2xKI mice, like those of humans, exhibit regional differences of vulnerability, with the hippocampus exhibiting the first signatures of structural changes that, in turn, may underlie the emergent inability to update spatial memory in later months.


Assuntos
Doença de Alzheimer/patologia , Espinhas Dendríticas/patologia , Córtex Entorrinal/patologia , Hipocampo/patologia , Neuropeptídeos/biossíntese , Doença de Alzheimer/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Córtex Entorrinal/metabolismo , Hipocampo/metabolismo , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Mutantes , Sinapses/metabolismo , Sinapses/patologia , Fatores de Tempo
4.
J Neurosci ; 27(11): 2815-24, 2007 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-17360903

RESUMO

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1beta are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1beta with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Terminações Pré-Sinápticas/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Células Cultivadas , Galinhas , Hipocampo/química , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Terminações Pré-Sinápticas/química , Ratos
5.
Proc Natl Acad Sci U S A ; 103(9): 3410-5, 2006 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-16492745

RESUMO

It is widely thought that Alzheimer's disease (AD) begins as a malfunction of synapses, eventually leading to cognitive impairment and dementia. Homeostatic synaptic scaling is a mechanism that could be crucial at the onset of AD but has not been examined experimentally. In this process, the synaptic strength of a neuron is modified so that the overall excitability of the cell is maintained. Here, we investigate whether synaptic scaling mediated by l-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2 x KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2 x KI mice exhibit age-related downscaling of AMPAR-mediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2 x KI mice show age-related deficits in bidirectional plasticity (long-term potentiation and long-term depression) and memory flexibility. These results suggest that AMPARs are important synaptic targets for AD and provide evidence that cognitive impairment may involve downscaling of postsynaptic AMPAR function.


Assuntos
Envelhecimento/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Regulação para Baixo , Receptores de AMPA/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Eletrofisiologia , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Memória , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Plasticidade Neuronal , Receptores de AMPA/genética , Sinapses/metabolismo , Sinapses/patologia
6.
Brain Res ; 1064(1-2): 66-74, 2005 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-16325786

RESUMO

Post-mortem cortices from patients diagnosed with Alzheimer's disease (AD) exhibit reduced levels of drebrin, an F-actin binding protein of dendritic spines and shafts. We used a mouse model of familial AD (FAD) to determine whether the density of cortical spines engaged in asymmetric (presumably excitatory) synapses and containing drebrin A is reduced and if so, whether this occurs prior to the emergence of beta amyloid deposits, when only soluble beta amyloid (Abeta) is present. Quantitative electron microscopic immunocytochemistry revealed that by 6 months, the proportion of postsynaptic spines with drebrin A within somatosensory cortex layer I was smaller for the FAD model mice, when compared to the corresponding region of WT mice (P < 0.0005). However, the areal density of postsynaptic spines containing drebrin A was relatively constant from 3 to 18 months and beyond for both genotypes, suggesting that drebrin A confers stability to postsynaptic spines. Further measurements confirmed that the reduced proportion of drebrin A-containing spines in brains of FAD mice at 6 months is due to the greater size and areal density of spine profiles lacking drebrin A. Thus, soluble Abeta could affect spines lacking drebrin A more strongly than spines containing drebrin A. At 6 months and older, a larger fraction of spinous drebrin A in 2xKI mice was located near the synaptic membrane, as compared to those of WT mice. This pattern may reflect an altered trafficking of synaptic molecules within spines, a factor adding to the decline of synaptic function and plasticity.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas de Membrana/metabolismo , Neuropeptídeos/metabolismo , Córtex Somatossensorial/metabolismo , Fatores Etários , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Modelos Animais de Doenças , Masculino , Análise por Pareamento , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Mutação , Presenilina-1 , Córtex Somatossensorial/citologia , Sinapses/metabolismo
7.
J Comp Neurol ; 483(4): 383-402, 2005 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-15700273

RESUMO

Drebrin A is a neuron-specific, actin binding protein. Evidence to date is from in vitro studies, consistently supporting the involvement of drebrin A in spinogenesis and synaptogenesis. We sought to determine whether drebrin A arrives at the plasma membrane of neurons, in vivo, in time to orchestrate spinogenesis and synaptogenesis. To this end, a new antibody was used to locate drebrin A in relation to electron microscopically imaged synapses during early postnatal days. Western blotting showed that drebrin A emerges at postnatal day (PNd) 6 and becomes progressively more associated with F-actin in the pellet fraction. Light microscopy showed high concentrations of drebrin A in the synaptic layers of the hippocampus and cortex. Electron microscopy revealed that drebrin A in these regions is located exclusively in dendrites both neonatally and in adulthood. In adulthood, nearly all of the synaptic drebrin A is within spines forming asymmetric excitatory synapses, verified by gamma-aminobutyric acid (GABA) negativity. At PNd7, patches of drebrin A immunoreactivity were discretely localized to the submembranous surfaces of dendrites forming slight protrusions-protospines. The drebrin A sites exhibited only thin postsynaptic densities and lacked axonal associations or were contacted by axons that contained only a few vesicles. Yet, because of their immunoreactivity to the NR2B subunit of N-methyl-D-aspartate receptors and immunonegativity of axon terminals to GABA, these could be presumed to be nascent, excitatory synapses. Thus, drebrin A may be involved in organizing the dendritic pool of actin for the formation of spines and of axospinous excitatory synapses during early postnatal periods.


Assuntos
Encéfalo/metabolismo , Dendritos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neuropeptídeos/metabolismo , Sinapses/metabolismo , Membranas Sinápticas/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Imuno-Histoquímica/métodos , Microscopia Imunoeletrônica/métodos , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/classificação , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/ultraestrutura , Membranas Sinápticas/ultraestrutura , Ácido gama-Aminobutírico/metabolismo
8.
J Neurosci ; 24(5): 1136-48, 2004 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-14762132

RESUMO

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3'-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14-29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.


Assuntos
Transportador 2 de Aminoácido Excitatório/biossíntese , Hipocampo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Especificidade de Anticorpos , Química Encefálica , Transportador 2 de Aminoácido Excitatório/genética , Heterozigoto , Hipocampo/citologia , Hipocampo/ultraestrutura , Homozigoto , Imuno-Histoquímica , Hibridização In Situ/métodos , Camundongos , Camundongos Knockout , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Transporte Proteico/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley
9.
J Neurosci ; 22(6): 2142-52, 2002 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11896154

RESUMO

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.


Assuntos
Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/biossíntese , Transportador 2 de Aminoácido Excitatório/genética , Neurônios/metabolismo , Prosencéfalo/metabolismo , Processamento Alternativo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Bucladesina/farmacologia , Células COS , Células Cultivadas , Clonagem Molecular , Dendritos/metabolismo , Dendritos/ultraestrutura , Biblioteca Gênica , Imuno-Histoquímica , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Prosencéfalo/citologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Transfecção , Regulação para Cima/efeitos dos fármacos
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