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1.
J Neurosci Methods ; 406: 110137, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38626853

RESUMO

BACKGROUND: The neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD). NEW METHOD: Postmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. RESULTS: Neuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: We optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.


Assuntos
Doença de Alzheimer , Separação Celular , Córtex Cerebral , Vesículas Extracelulares , Doença de Alzheimer/patologia , Vesículas Extracelulares/patologia , Separação Celular/métodos , Córtex Cerebral/patologia , Humanos , Criopreservação , Autopsia , RNA-Seq , Neuroglia/patologia , Neurônios/patologia
2.
J Neurosci ; 43(26): 4941-4954, 2023 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-37253603

RESUMO

Synaptic loss is intrinsically linked to Alzheimer's disease (AD) neuropathology and symptoms, but its direct impact on clinical symptoms remains elusive. The postsynaptic protein Shank3 (SH3 and multiple ankyrin repeat domains) is of particular interest, as the loss of a single allele of the SHANK3 gene is sufficient to cause profound cognitive symptoms in children. We thus sought to determine whether a SHANK3 deficiency could contribute to the emergence or worsening of AD symptoms and neuropathology. We first found a 30%-50% postmortem loss of SHANK3a associated with cognitive decline in the parietal cortex of individuals with AD. To further probe the role of SHANK3 in AD, we crossed male and female 3xTg-AD mice modelling Aß and tau pathologies with Shank3a-deficient mice (Shank3Δex4-9). We observed synergistic deleterious effects of Shank3a deficiency and AD neuropathology on object recognition memory at 9, 12, and 18 months of age and on anxious behavior at 9 and 12 months of age in hemizygous Shank3Δex4-9-3xTg-AD mice. In addition to the expected 50% loss of Shank3a, levels of other synaptic proteins, such as PSD-95, drebrin, and homer1, remained unchanged in the parietotemporal cortex of hemizygous Shank3Δex4-9 animals. However, Shank3a deficiency increased the levels of soluble Aß42 and human tau at 18 months of age compared with 3xTg-AD mice with normal Shank3 expression. The results of this study in human brain samples and in transgenic mice are consistent with the hypothesis that Shank3 deficiency makes a key contribution to cognitive impairment in AD.SIGNIFICANCE STATEMENT Although the loss of several synaptic proteins has been described in Alzheimer's disease (AD), it remains unclear whether their reduction contributes to clinical symptoms. The results of this study in human samples show lower levels of SHANK3a in AD brain, correlating with cognitive decline. Data gathered in a novel transgenic mouse suggest that Shank3a deficiency synergizes with AD neuropathology to induce cognitive impairment, consistent with a causal role in AD. Therefore, treatment aiming at preserving Shank3 in the aging brain may be beneficial to prevent AD.


Assuntos
Doença de Alzheimer , Animais , Feminino , Humanos , Masculino , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Cognição , Modelos Animais de Doenças , Camundongos Transgênicos , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Proteínas tau/genética , Proteínas tau/metabolismo
3.
Front Pharmacol ; 12: 766082, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34925024

RESUMO

Alzheimer's disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aß. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

5.
Acta Neuropathol ; 142(3): 495-511, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33991233

RESUMO

The diagnosis of Parkinson's disease (PD) and atypical parkinsonian syndromes is difficult due to the lack of reliable, easily accessible biomarkers. Multiple system atrophy (MSA) is a synucleinopathy whose symptoms often overlap with PD. Exosomes isolated from blood by immunoprecipitation using CNS markers provide a window into the brain's biochemistry and may assist in distinguishing between PD and MSA. Thus, we asked whether α-synuclein (α-syn) in such exosomes could distinguish among healthy individuals, patients with PD, and patients with MSA. We isolated exosomes from the serum or plasma of these three groups by immunoprecipitation using neuronal and oligodendroglial markers in two independent cohorts and measured α-syn in these exosomes using an electrochemiluminescence ELISA. In both cohorts, α-syn concentrations were significantly lower in the control group and significantly higher in the MSA group compared to the PD group. The ratio between α-syn concentrations in putative oligodendroglial exosomes compared to putative neuronal exosomes was a particularly sensitive biomarker for distinguishing between PD and MSA. Combining this ratio with the α-syn concentration itself and the total exosome concentration, a multinomial logistic model trained on the discovery cohort separated PD from MSA with an AUC = 0.902, corresponding to 89.8% sensitivity and 86.0% specificity when applied to the independent validation cohort. The data demonstrate that a minimally invasive blood test measuring α-syn in blood exosomes immunoprecipitated using CNS markers can distinguish between patients with PD and patients with MSA with high sensitivity and specificity. Future optimization and validation of the data by other groups would allow this strategy to become a viable diagnostic test for synucleinopathies.


Assuntos
Exossomos/imunologia , Atrofia de Múltiplos Sistemas/diagnóstico , Neurônios/metabolismo , Oligodendroglia/metabolismo , Doença de Parkinson/diagnóstico , alfa-Sinucleína/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores , Estudos de Coortes , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Feminino , Voluntários Saudáveis , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/sangue , Doença de Parkinson/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
ACS Chem Biol ; 15(6): 1671-1684, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32352753

RESUMO

We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo. Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.


Assuntos
Acetilcolinesterase/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Inibidores Enzimáticos/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Inibidores da Colinesterase/química , Inibidores da Colinesterase/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Relação Estrutura-Atividade
7.
Alzheimer Dis Assoc Disord ; 33(4): 327-330, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31513029

RESUMO

OBJECTIVE: A rare variant in TREM2 (p.R47H, rs75932628) has been consistently reported to increase the risk for Alzheimer disease (AD), while mixed evidence has been reported for association of the variant with other neurodegenerative diseases. Here, we investigated the frequency of the R47H variant in a diverse and well-characterized multicenter neurodegenerative disease cohort. METHODS: We examined the frequency of the R47H variant in a diverse neurodegenerative disease cohort, including a total of 3058 patients clinically diagnosed with AD, frontotemporal dementia spectrum syndromes, mild cognitive impairment, progressive supranuclear palsy syndrome, corticobasal syndrome, or amyotrophic lateral sclerosis and 5089 control subjects. RESULTS: We observed a significant association between the R47H variant and AD, while no association was observed with any other neurodegenerative disease included in this study. CONCLUSIONS: Our results support the consensus that the R47H variant is significantly associated with AD. However, we did not find evidence for association of the R47H variant with other neurodegenerative diseases.


Assuntos
Predisposição Genética para Doença , Variação Genética , Genótipo , Glicoproteínas de Membrana/genética , Doenças Neurodegenerativas/genética , Receptores Imunológicos/genética , Idoso , Doença de Alzheimer/genética , Esclerose Lateral Amiotrófica/genética , Disfunção Cognitiva/genética , Estudos de Coortes , Feminino , Demência Frontotemporal/genética , Humanos , Internacionalidade , Masculino
8.
Am J Pathol ; 189(8): 1621-1636, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31108099

RESUMO

Apolipoprotein E (apoE) colocalizes with amyloid-ß (Aß) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aß are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aß complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aß complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aß was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aß concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aß was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aß complex and associated lipids into synaptic terminals, with subsequent Aß clearance in control synapses and accumulation in AD synapses.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/metabolismo , Córtex Cerebral/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/metabolismo , Sinapses/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Córtex Cerebral/patologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sinapses/patologia , Sinaptossomos/metabolismo , Sinaptossomos/patologia
9.
Neurobiol Dis ; 114: 120-128, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29501530

RESUMO

Synaptic neurodegeneration is thought to be an early event initiated by soluble ß-amyloid (Aß) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aß aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aß oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7-8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4+/+/FAD+/-) relative to E4FAD- (non-carrier; APOE4+/+/FAD-/-) mice, suggesting NP1 is modulated by Aß expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD.


Assuntos
Doença de Alzheimer/sangue , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/sangue , Sinapses/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Animais , Biomarcadores/sangue , Encéfalo/patologia , Proteína C-Reativa , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Sinapses/patologia
10.
Neuron ; 94(2): 278-293.e9, 2017 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-28426964

RESUMO

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aß fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo
11.
Neurol Clin ; 35(2): 175-190, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28410655

RESUMO

To date, Alzheimer disease drug candidates have produced negative results in human trials, and progress in moving new targets out of the laboratory and into trials has been slow. However, based on 3 decades of previous work, there is reason to hope that amyloid-based and other novel therapies will move at a faster pace toward successful clinical trials. This article highlights selected preclinical research topics that are rapidly advancing in the laboratory.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apolipoproteínas E/genética , Inflamação/complicações , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Colesterol/metabolismo , Humanos
12.
J Neurosci ; 37(5): 1197-1212, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27986924

RESUMO

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.


Assuntos
Doença de Alzheimer/fisiopatologia , Potenciação de Longa Duração/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Estimulação Elétrica , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Fosfodiesterase/farmacologia , Ratos Sprague-Dawley , Receptores de AMPA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos
13.
Sci Rep ; 6: 18725, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26729583

RESUMO

Altered brain metabolism is associated with progression of Alzheimer's Disease (AD). Mitochondria respond to bioenergetic changes by continuous fission and fusion. To account for three dimensional architecture of the brain tissue and organelles, we applied 3-dimensional electron microscopy (3D EM) reconstruction to visualize mitochondrial structure in the brain tissue from patients and mouse models of AD. We identified a previously unknown mitochondrial fission arrest phenotype that results in elongated interconnected organelles, "mitochondria-on-a-string" (MOAS). Our data suggest that MOAS formation may occur at the final stages of fission process and was not associated with altered translocation of activated dynamin related protein 1 (Drp1) to mitochondria but with reduced GTPase activity. Since MOAS formation was also observed in the brain tissue of wild-type mice in response to hypoxia or during chronological aging, fission arrest may represent fundamental compensatory adaptation to bioenergetic stress providing protection against mitophagy that may preserve residual mitochondrial function. The discovery of novel mitochondrial phenotype that occurs in the brain tissue in response to energetic stress accurately detected only using 3D EM reconstruction argues for a major role of mitochondrial dynamics in regulating neuronal survival.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/ultraestrutura , Modelos Animais de Doenças , Dinaminas/metabolismo , Hipóxia/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Fenótipo , Fosforilação
14.
Am J Pathol ; 186(1): 185-98, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26718979

RESUMO

Amyloid-ß (Aß) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aß and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aß and p-tau in large populations of individual synaptic terminals. Soluble Aß oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aß was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aß oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aß-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aß drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Sinapses/patologia , Proteínas tau/análise , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia Confocal , Fosforilação , Ratos , Ratos Transgênicos
15.
Alzheimers Dement (Amst) ; 1(2): 187-193, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26207261

RESUMO

BACKGROUND: Brain-derived neurotrophic factor (BDNF) plays an important role in Alzheimer's disease (AD) and other neurodegenerative disorders. BDNF function is adversely affected by amyloid beta (Aß) in AD. BDNF levels in brain and peripheral tissues are lower in patients with AD and MCI, than in controls. Here we examined the association between plasma levels of BDNF and amyloid deposition in the brain measured with Pittsburgh Compound B (PiB). METHOD: Our dataset consisted of 18 AD, 56 mild cognitive impairment (MCI) and 3 normal control (NC) Alzheimer's Disease Neuroimaging Initiative-1 (ADNI1) subjects with available [11C] PiB and peripheral blood protein data. MRI-coregistered PET data was smoothed with a 15 mm kernel and mapped onto 3D hemispheric models using the warping deformations computed in cortical pattern matching of the associated MRI scans. We applied linear regression to examine in 3D the associations between BDNF and PiB SUVR, while adjusting for age and sex. We used permutation statistics thresholded at p<0.01 for multiple comparisons correction. RESULTS: Plasma BDNF levels showed significant negative associations with left greater than right amyloid burden in the lateral temporal, inferior parietal, inferior frontal, anterior and posterior cingulate, and orbitofrontal regions (left pcorrected=0.03). CONCLUSIONS: As hypothesized, lower plasma levels of BDNF were significantly associated with widespread brain amyloidosis.

16.
Dement Geriatr Cogn Disord ; 39(3-4): 154-66, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25547651

RESUMO

Apolipoprotein E (APOE) genotype is the strongest known genetic risk factor for sporadic Alzheimer's disease (AD), but the utility of plasma ApoE levels for assessing the severity of underlying neurodegenerative changes remains uncertain. Here, we examined cross-sectional associations between plasma ApoE levels and volumetric magnetic resonance imaging indices of the hippocampus from 541 participants [57 with normal cognition (NC), 375 with mild cognitive impairment (MCI), and 109 with mild AD] who were enrolled in the Alzheimer's Disease Neuroimaging Initiative. Across the NC and MCI groups, lower plasma ApoE levels were significantly correlated with smaller hippocampal size, as measured by either hippocampal volume or hippocampal radial distance. These associations were driven primarily by findings from carriers of an APOE ε4 allele and are consistent with prior reports that lower plasma ApoE levels correlate with greater global cortical Pittsburgh Compound B retention. In this high-risk group, plasma ApoE levels may represent a peripheral marker of underlying AD neuropathology in nondemented elderly individuals.


Assuntos
Doença de Alzheimer/sangue , Apolipoproteína E4/sangue , Disfunção Cognitiva/genética , Hipocampo/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Apolipoproteína E4/genética , Biomarcadores/sangue , Estudos Transversais , Feminino , Genótipo , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Neuroimagem/métodos , Testes Neuropsicológicos , Tamanho do Órgão
17.
J Neurochem ; 133(3): 368-79, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25393609

RESUMO

The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from pre-synaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the pre-synaptic compartment in AD. Results demonstrate the abundance of tau, mainly C-terminal truncated tau, in synaptic terminals in aged control and in Alzheimer's disease (AD) samples. Tau fragments and dimers/oligomers are prominent in AD synapses. Following depolarization, tau release is potentiated in AD nerve terminals compared to aged controls. We hypothesize (i) endosomal release of the different tau peptides from AD synapses, and (ii) together with phosphorylation, fragmentation of synaptic tau exacerbates tau aggregation, synaptic dysfunction, and the spread of tau pathology in AD. Aß = amyloid-beta.


Assuntos
Doença de Alzheimer/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
Alzheimers Res Ther ; 4(5): 43, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23107780

RESUMO

INTRODUCTION: Curcumin is a polyphenolic compound derived from the plant Curcuma Long Lin that has been demonstrated to have antioxidant and anti-inflammatory effects as well as effects on reducing beta-amyloid aggregation. It reduces pathology in transgenic models of Alzheimer's disease (AD) and is a promising candidate for treating human AD. The purpose of the current study is to generate tolerability and preliminary clinical and biomarker efficacy data on curcumin in persons with AD. METHODS: We performed a 24-week randomized, double blind, placebo-controlled study of Curcumin C3 Complex(®) with an open-label extension to 48 weeks. Thirty-six persons with mild-to-moderate AD were randomized to receive placebo, 2 grams/day, or 4 grams/day of oral curcumin for 24 weeks. For weeks 24 through 48, subjects that were receiving curcumin continued with the same dose, while subjects previously receiving placebo were randomized in a 1:1 ratio to 2 grams/day or 4 grams/day. The primary outcome measures were incidence of adverse events, changes in clinical laboratory tests and the Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-Cog) at 24 weeks in those completing the study. Secondary outcome measures included the Neuropsychiatric Inventory (NPI), the Alzheimer's Disease Cooperative Study - Activities of Daily Living (ADCS-ADL) scale, levels of Aß1-40 and Aß1-42 in plasma and levels of Aß1-42, t-tau, p-tau181 and F2-isoprostanes in cerebrospinal fluid. Plasma levels of curcumin and its metabolites up to four hours after drug administration were also measured. RESULTS: Mean age of completers (n = 30) was 73.5 years and mean Mini-Mental Status Examination (MMSE) score was 22.5. One subject withdrew in the placebo (8%, worsened memory) and 5/24 subjects withdrew in the curcumin group (21%, 3 due to gastrointestinal symptoms). Curcumin C3 Complex(®) was associated with lowered hematocrit and increased glucose levels that were clinically insignificant. There were no differences between treatment groups in clinical or biomarker efficacy measures. The levels of native curcumin measured in plasma were low (7.32 ng/mL). CONCLUSIONS: Curcumin was generally well-tolerated although three subjects on curcumin withdrew due to gastrointestinal symptoms. We were unable to demonstrate clinical or biochemical evidence of efficacy of Curcumin C3 Complex(®) in AD in this 24-week placebo-controlled trial although preliminary data suggest limited bioavailability of this compound. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00099710.

19.
Arch Neurol ; 69(6): 757-64, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22689192

RESUMO

OBJECTIVE: To study the effect of familial Alzheimer disease (FAD) mutations and APOE genotype on plasma signaling protein levels. DESIGN: Cross-sectional comparison of plasma levels of 77 proteins measured using multiplex immune assays. SETTING: A tertiary referral dementia research center. PARTICIPANTS: Thirty-three persons from families harboring PSEN1 or APP mutations, aged 19 to 59 years. MAIN OUTCOME MEASURES: Protein levels were compared between FAD mutation carriers (MCs) and noncarriers (NCs) and among APOE genotype groups, using multiple linear regression models. RESULTS: Twenty-one participants were FAD MCs and 12 were NCs. Six had the APOE ε2/3, 6 had the ε3/4, and 21 had the ε3/3 genotype. Levels of 17 proteins differed among APOE genotype groups, and there were significant interactions between age and APOE genotype for 12 proteins. Plasma levels of apolipoprotein E and superoxide dismutase 1 were highest in the ε2 carriers, lowest in ε4 carriers, and intermediate in the ε3 carriers. Levels of multiple interleukins showed the opposite pattern and, among the ε4 carriers, demonstrated significant negative correlations with age. Although there were no significant differences between FAD MCs and NCs, there were interactions between mutation status and APOE genotype for 13 proteins. CONCLUSIONS: We found different patterns of inflammatory markers in young and middle-aged persons among APOE genotype groups. The APOE ε4 carriers had the lowest levels of apolipoprotein E. Young ε4 carriers have increased inflammatory markers that diminish with age. We demonstrated altered inflammatory responses in young and middle adulthood in ε4 carriers that may relate to AD risk later in life.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Proteínas Sanguíneas/metabolismo , Mutação , Adulto , Fatores Etários , Precursor de Proteína beta-Amiloide/genética , Citocinas/metabolismo , Feminino , Testes Genéticos , Genótipo , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Presenilina-1/genética , Escalas de Graduação Psiquiátrica , Fatores de Risco , Adulto Jovem
20.
Cytometry A ; 81(3): 248-54, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22213704

RESUMO

Amyloid beta (Aß) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aß, but the Aß and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aß-positive synaptosomes with the goal of understanding the nature of Aß and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aß were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aß-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aß oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aß and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Córtex Cerebral/patologia , Terminações Pré-Sinápticas/patologia , Sinaptossomos/patologia , Proteínas tau/análise , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Animais , Córtex Cerebral/química , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Camundongos , Microscopia Eletrônica/métodos , Sinaptossomos/fisiologia , Proteínas tau/química
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