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1.
Can J Neurol Sci ; 46(5): 499-511, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31309917

RESUMO

BACKGROUND: The Comprehensive Assessment of Neurodegeneration and Dementia (COMPASS-ND) cohort study of the Canadian Consortium on Neurodegeneration in Aging (CCNA) is a national initiative to catalyze research on dementia, set up to support the research agendas of CCNA teams. This cross-country longitudinal cohort of 2310 deeply phenotyped subjects with various forms of dementia and mild memory loss or concerns, along with cognitively intact elderly subjects, will test hypotheses generated by these teams. METHODS: The COMPASS-ND protocol, initial grant proposal for funding, fifth semi-annual CCNA Progress Report submitted to the Canadian Institutes of Health Research December 2017, and other documents supplemented by modifications made and lessons learned after implementation were used by the authors to create the description of the study provided here. RESULTS: The CCNA COMPASS-ND cohort includes participants from across Canada with various cognitive conditions associated with or at risk of neurodegenerative diseases. They will undergo a wide range of experimental, clinical, imaging, and genetic investigation to specifically address the causes, diagnosis, treatment, and prevention of these conditions in the aging population. Data derived from clinical and cognitive assessments, biospecimens, brain imaging, genetics, and brain donations will be used to test hypotheses generated by CCNA research teams and other Canadian researchers. The study is the most comprehensive and ambitious Canadian study of dementia. Initial data posting occurred in 2018, with the full cohort to be accrued by 2020. CONCLUSION: Availability of data from the COMPASS-ND study will provide a major stimulus for dementia research in Canada in the coming years.


Évaluation complète d'une étude de cohorte canadienne portant sur la démence et la neuro-dégénérescence. Contexte : L'évaluation globale de la neuro-dégénérescence et de la démence (COMPASS-ND), étude de cohorte du Consortium canadien en neuro-dégénérescence associée au vieillissement (CCNV), représente une initiative nationale visant à promouvoir la recherche portant sur la démence et à soutenir les programmes de recherche des équipes du CCNV. Totalisant 2310 sujets recrutés partout au pays, cette cohorte longitudinale regroupe des individus fortement « phénotypés ¼ qui présentent diverses formes de démence et de pertes de mémoire légères. En plus de sujets âgés dont les fonctions cognitives sont intactes, ces 2310 sujets ont permis de valider les hypothèses formulées par les équipes du CCNV. Méthodes : Nous avons utilisé de nombreux documents pour décrire cette étude : le protocole de la COMPASS-ND ; la demande initiale de subvention ; le cinquième rapport d'étape semi-annuel du CCNV soumis aux Instituts de recherche en santé du Canada (IRSC) en décembre 2017 ; ainsi que d'autres documents produits à la suite de modifications consécutives à la mise en œuvre de ce projet. Résultats: L'étude de cohorte COMPASS-ND du CCNV inclut des participants de partout au Canada dont les divers états cognitifs sont associés à des maladies neurodégénératives ou au risque d'en souffrir. Ils feront l'objet d'un large éventail d'examens expérimentaux, cliniques, génétiques et d'imagerie afin d'aborder de manière spécifique les causes, le diagnostic, le traitement et la prévention de ces états cognitifs chez les personnes âgées. Les données obtenues à la suite d'évaluations cliniques et cognitives, ainsi que celles issues d'échantillons biologiques, d'imagerie cérébrale, de tests génétiques et de dons de cerveaux, seront utilisées pour tester les hypothèses générées par les équipes de recherche du CCNV et d'autres chercheurs canadiens. Cette étude constitue donc à ce jour l'étude canadienne la plus complète et la plus ambitieuse au sujet de la démence. La présentation des données initiales ayant eu lieu en 2018, la cohorte devrait atteindre sa taille maximale d'ici à 2020.Conclusion : La disponibilité des données de l'étude COMPASS-ND stimulera considérablement la recherche sur la démence au Canada au cours des prochaines années.


Assuntos
Envelhecimento , Demência , Doenças Neurodegenerativas , Projetos de Pesquisa , Canadá , Estudos de Coortes , Feminino , Humanos , Estudos Longitudinais , Masculino
2.
J Neurochem ; 137(4): 630-46, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26871972

RESUMO

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.


Assuntos
Colina O-Acetiltransferase/metabolismo , Mutação/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinação/fisiologia , Animais , Catálise , Células Cultivadas , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/metabolismo , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/genética
3.
Neurobiol Dis ; 69: 32-42, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24844149

RESUMO

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to ß-amyloid peptides (Aß), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aß production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aß1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aß1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aß production. This decreased formation of Aß could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aß. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aß production, with this potentiating the decline in function of cholinergic neurons.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Colina O-Acetiltransferase/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Neurônios Colinérgicos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Camundongos Transgênicos , Análise em Microsséries , Presenilina-1/genética , Presenilina-1/metabolismo , Regiões Promotoras Genéticas
4.
J Neurosci ; 32(16): 5573-84, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22514319

RESUMO

Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO⁻) donor]; ONOO⁻ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamin-dependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle- and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.


Assuntos
Colina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Molsidomina/análogos & derivados , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Clatrina/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Hemicolínio 3/farmacocinética , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Molsidomina/farmacologia , Mutação/genética , Neuroblastoma/patologia , Ácido Peroxinitroso/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Fatores de Tempo , Transfecção , Trítio/metabolismo , Trítio/farmacocinética , Ubiquitinação/fisiologia , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
5.
J Biol Chem ; 287(44): 37245-58, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22948140

RESUMO

We previously demonstrated that nerve cell lines selected for resistance to amyloid ß (Aß) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aß and other neurotoxins. Treatment of Aß-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aß treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aß or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αß or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aß deposition without developing Alzheimer disease.


Assuntos
Peptídeos beta-Amiloides/fisiologia , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Animais , Estudos de Casos e Controles , Linhagem Celular , Respiração Celular , Córtex Cerebral/enzimologia , Feminino , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5 , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Transgênicos , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Estaurosporina/farmacologia
6.
J Biol Chem ; 285(47): 36542-50, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20837487

RESUMO

The prion protein (PrP(C)) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP(C)-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C)·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.


Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Choque Térmico/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas PrPC/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Choque Térmico/genética , Hipocampo/citologia , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Ligação Proteica , RNA Mensageiro/genética , Receptores Nicotínicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Nicotínico de Acetilcolina alfa7
7.
J Neurochem ; 117(3): 538-53, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21352228

RESUMO

In Alzheimer's disease, the amyloid-ß peptide (Aß) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aß. We show here that Aß oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aß oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aß oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aß oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aß oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aß oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aß oligomers. Our experiments show for the first time that Aß oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/metabolismo , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas PrPC/metabolismo , Análise de Variância , Animais , Biotinilação/métodos , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Camundongos , Microscopia Confocal/métodos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Transporte Proteico/efeitos dos fármacos , Fatores de Tempo , Transfecção , Proteínas rab5 de Ligação ao GTP/metabolismo
8.
Nat Cell Biol ; 4(8): 547-55, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12105416

RESUMO

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.


Assuntos
Arrestinas/metabolismo , Citoesqueleto/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Animais , Arrestinas/química , Transporte Biológico Ativo , Células COS , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Biológicos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , beta-Arrestinas , Proteínas ral de Ligação ao GTP/química , Fator ral de Troca do Nucleotídeo Guanina/química
9.
Front Cell Dev Biol ; 9: 722560, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557490

RESUMO

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

10.
Can J Aging ; 39(4): 506-512, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32867867

RESUMO

Cet article présente les domaines prioritaires de recherche sur les impacts de la pandémie de COVID-19 chez les personnes âgées telles qu'ils ont été identifiés par l'Institut du vieillissement des IRSC (IV-IRSC). Le processus utilisé par l'IV-IRSC a comporté plusieurs phases itératives qui ont permis d'identifier trois secteurs prioritaires parmi les besoins de la recherche relative à la COVID-19, et quatre axes thématiques transversaux. Les secteurs de recherche prioritaires sont : 1) la réponse des personnes âgées à la maladie, à la vaccination et aux traitements, 2) la santé mentale et l'isolement, et 3) les milieux de soins soutenants. Les quatre thèmes transversaux sont : a) l'Équité, la diversité et l'inclusion (EDI), b) les considérations éthiques et morales, c) les pratiques fondées sur les données probantes, et d) les technologies numériques de la santé. Les priorités décrites dans cet article guideront les réponses de l'IV-IRSC aux besoins de la recherche sur la COVID-19.

11.
J Neuropathol Exp Neurol ; 79(11): 1147-1162, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33011810

RESUMO

ß-Amyloid (Aß) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMϕ) from hematogenous macrophages (hMϕ), as they are morphologically and phenotypically similar when activated. To distinguish between mMϕ and hMϕ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMϕ, but not in mMϕ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMϕ from hMϕ. Mutant mice demonstrated robust increases in Aß1-42, area of Aß plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aß accumulation, paralleled by the accumulation of hMϕ around Aß plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aß plaques was age- and sex-dependent and preceded cognitive impairment.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Macrófagos/patologia , Placa Amiloide/patologia , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/imunologia , Modelos Animais de Doenças , Feminino , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , Placa Amiloide/imunologia
12.
Pediatr Res ; 65(3): 301-6, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19033882

RESUMO

Reduced cerebral function after neonatal hypoxia-ischemia is an early indicator of hypoxic-ischemic encephalopathy. Near-infrared spectroscopy offers a clinically relevant means of detecting impaired cerebral metabolism from the measurement of the cerebral metabolic rate of oxygen (CMRO2). The purpose of this study was to determine the relationship between postinsult CMRO2 and duration of hypoxia-ischemia in piglets. Twelve piglets were subjected to randomly selected durations of hypoxia-ischemia (5-28 min) and five animals served as controls. Measurements of CMRO2 were taken before and for 24 h after hypoxia-ischemia. Histology was carried out in nine piglets (six insults, three controls) to estimate brain injury. In the first 4 h after the insult, average CMRO2 of the insult group was significantly depressed (33 +/- 3% reduction compared with controls) and by 8 h, a significant correlation developed, which persisted for the remainder of the study, between CMRO2 and the duration of ischemia. Histologic staining suggested little brain damage resulted from shorter insult durations and considerable damage from more prolonged insults. This study demonstrated that near-infrared spectroscopy could detect early changes in CMRO2 after hypoxia-ischemia for a range of insult severities and CMRO2 could be used to distinguish insult severity by 8 h after the insult.


Assuntos
Cérebro/metabolismo , Hipóxia-Isquemia Encefálica/diagnóstico , Hipóxia-Isquemia Encefálica/metabolismo , Oxigênio/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Cérebro/patologia , Sus scrofa , Fatores de Tempo
13.
Mol Pharmacol ; 73(3): 801-12, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17971421

RESUMO

Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Ácido Peroxinitroso/metabolismo , Sódio/metabolismo , Animais , Biotinilação , Técnicas de Cultura de Células , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Colina/antagonistas & inibidores , Colina/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacologia , Meios de Cultura , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hemicolínio 3/metabolismo , Hemicolínio 3/farmacologia , Humanos , Concentração Inibidora 50 , Rim/citologia , Cinética , L-Lactato Desidrogenase/análise , Luminescência , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Neuroblastoma/patologia , Nitrogênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/biossíntese , Transporte Proteico , Ratos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transfecção , Tirosina/metabolismo
14.
Front Mol Neurosci ; 11: 60, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29541020

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons. Dysregulation of epigenetic processes have been observed in the aging brain and in patients with AD, though there is not yet agreement on the impact of these changes on transcription. New evidence shows that proteins involved in genome organization have altered expression and localization in the AD brain, suggesting that the genomic landscape may play a critical role in the development of AD. This review discusses the role of the chromatin organizers and epigenetic modifiers in post-mitotic cells, the aging brain, and in the development and progression of AD. How these new insights can be used to help determine disease risk and inform treatment strategies will also be discussed.

15.
Eur J Neurosci ; 26(12): 3437-48, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18088276

RESUMO

The high-affinity choline transporter (CHT1) is responsible for uptake of choline from the synaptic cleft and supplying choline for acetylcholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K(+)-depolarization promotes Ca(2+)-dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K(+)-depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular transporters to the recycling pool.


Assuntos
Membrana Celular/metabolismo , Simportadores/metabolismo , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Endossomos/metabolismo , Imunofluorescência/métodos , Humanos , Immunoblotting , Cloreto de Potássio/farmacologia , Coloração e Rotulagem , Simportadores/genética , Fatores de Tempo , Transfecção
16.
Front Mol Neurosci ; 10: 361, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163036

RESUMO

Alzheimer's disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived ß-amyloid peptides (Aß) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aß peptides released from neural cells. The Aß-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA1). BafA1 also attenuated the Aß-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aß on CHT activity. Importantly, neutralizing Aß using an anti-Aß antibody directed at the N-terminal amino acids 1-16 of Aß, but not by an antibody directed at the mid-region amino acids 22-35 of Aß, attenuates the effect of Aß on CHT activity and trafficking. This indicates that a specific N-terminal Aß epitope, or specific conformation of soluble Aß, may impair CHT activity. Therefore, Aß immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.

17.
Front Mol Neurosci ; 10: 415, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29311808

RESUMO

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease.

18.
Alzheimers Dement (N Y) ; 3(4): 660-667, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29255793

RESUMO

INTRODUCTION: Despite important sex differences, there remains a paucity of studies examining sex and gender differences in neurodegeneration. The Canadian Consortium on Neurodegeneration in Aging (CCNA), a national network of researchers, provides an ideal platform to incorporate sex and gender. METHODS: CCNA's Women, Gender, Sex and Dementia program developed and implemented a six-component strategy involving executive oversight, training, research collaboration, progress report assessment, results dissemination, and ongoing manuscript review. The inclusion of sex and gender in current and planned CCNA projects was examined in two progress reporting periods in 2016. RESULTS: Sex and gender research productivity increased substantially for both preclinical (36%-45%) and human (56%-60%) cohorts. The main barrier was lack of funding. DISCUSSION: The Women, Gender, Sex and Dementia strategy resulted in a major increase of sex and gender into research on neurodegenerative disorders. This best practice model could be utilized by a wide variety of large multidisciplinary groups.

19.
Sci Rep ; 6: 23914, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27052102

RESUMO

The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-ß1-42 (Aß1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aß1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aß1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aß1-42-induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aß1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aß-exposure.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Colina O-Acetiltransferase/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Regiões de Interação com a Matriz/efeitos dos fármacos , Neurônios/citologia , Precursor de Proteína beta-Amiloide/genética , Linhagem Celular , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons/efeitos dos fármacos , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos
20.
J Neurosci Methods ; 148(1): 26-35, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16176837

RESUMO

Immunohistochemical and immunofluorescence staining approaches are powerful tools for characterization of the endogenous protein expression and subcellular compartmentalization. However, several technical problems hamper identification of low-abundance nuclear proteins in archival formalin-fixed, paraffin-embedded human neural tissue. These include loss of protein antigenicity during tissue fixation and processing, and intrinsic auto-fluorescence associated with the tissue related to its fixation and the presence of lipofuscin. We evaluated several antigen retrieval methods to establish a strategy for detection of neuronal nuclear proteins in human spinal cord formalin-fixed, paraffin-embedded tissue. Thus, using immunostaining of the neuron-specific nuclear protein NeuN as the outcome measure, we found that heating tissue sections in an alkaline pH buffer unmasked protein epitopes most effectively. Moreover, staining by immunohistochemistry with diaminobenzidine tetrahydrochloride chromagen was superior to immunofluorescence labeling, likely due to the signal amplification steps included in the former approach. Auto-fluorescence in the tissue sections can be effectively reduced, but a sufficient fluorescence signal associated with specific antibody labeling could not be detected above this background for NeuN in the nucleus.


Assuntos
Antígenos Nucleares/análise , Proteínas do Tecido Nervoso/análise , Inclusão em Parafina/métodos , Fosfopiruvato Hidratase/metabolismo , Medula Espinal/patologia , Fixação de Tecidos/métodos , Animais , Compostos Azo/farmacologia , Boratos/farmacologia , Corantes/farmacologia , Fixadores , Formaldeído , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica/métodos , Indóis/metabolismo , Camundongos , Naftalenos , Necrose , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo
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