RESUMO
Alzheimer's disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid ß-protein (Aß42) forms and novel intraneuronal Aß oligomers (AßOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aß uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aß42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aß pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aß42, far-peripheral AßOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.
Assuntos
Doença de Alzheimer , Masculino , Humanos , Feminino , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Proteoma/metabolismo , Proteômica , Retina/patologia , Atrofia/patologia , Biomarcadores/metabolismoRESUMO
The development of new therapeutic avenues that target the early stages of Alzheimer's disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-ß. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.
Assuntos
Doença de Alzheimer , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Endocitose , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Sinapses/metabolismoRESUMO
Microglia are the sentinels of the brain but a clear understanding of the factors that modulate their activation in physiological and pathological conditions is still lacking. Here we demonstrate that Nerve Growth Factor (NGF) acts on microglia by steering them toward a neuroprotective and anti-inflammatory phenotype. We show that microglial cells express functional NGF receptors in vitro and ex vivo. Our transcriptomic analysis reveals how, in primary microglia, NGF treatment leads to a modulation of motility, phagocytosis and degradation pathways. At the functional level, NGF induces an increase in membrane dynamics and macropinocytosis and, in vivo, it activates an outward rectifying current that appears to modulate glutamatergic neurotransmission in nearby neurons. Since microglia are supposed to be a major player in Aß peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA-mediated engulfment of Aß by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aß treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aß-induced loss of dendritic spines and inhibition of long term potentiation. Finally, in an ex-vivo setup of acute brain slices, we observed a similar increase in Aß engulfment by microglial cells under the influence of NGF. Our work substantiates a role for NGF in the regulation of microglial homeostatic activities and points toward this neurotrophin as a neuroprotective agent in Aß accumulation pathologies, via its anti-inflammatory activity on microglia.
Assuntos
Microglia/metabolismo , Fator de Crescimento Neural/metabolismo , Neuroproteção/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Células Cultivadas , Técnicas de Cocultura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Fator de Crescimento Neural/administração & dosagem , Neurônios/citologia , Neurônios/metabolismo , Fagocitose/fisiologia , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Transmissão Sináptica/fisiologia , Técnicas de Cultura de Tecidos , TranscriptomaRESUMO
Nerve growth factor is a therapeutic candidate for Alzheimer's disease. Due to its pain-inducing activity, in current clinical trials nerve growth factor is delivered locally into the brain by neurosurgery, but data on the efficacy of local nerve growth factor delivery in decreasing amyloid-ß deposition are not available. To reduce the nerve growth factor pain-inducing side effects, thus avoiding the need for local brain injection, we developed human painless nerve growth factor (hNGFp), inspired by the human genetic disease hereditary sensory and autonomic neuropathy type V. hNGFp has identical neurotrophic potency as wild-type human nerve growth factor, but a 10-fold lower pain sensitizing activity. In this study we first mimicked, in the 5xFAD mouse model, the intraparenchymal delivery of hNGFp used in clinical trials and found it to be ineffective in decreasing amyloid-ß plaque load. On the contrary, the same dose of hNGFp delivered intranasally, which was widely biodistributed in the brain and did not induce pain, showed a potent anti-amyloidogenic action and rescued synaptic plasticity and memory deficits. We found that hNGFp acts on glial cells, modulating inflammatory proteins such as the soluble TNFα receptor II and the chemokine CXCL12. We further established that the rescuing effect by hNGFp is mediated by CXCL12, as pharmacological inhibition of CXCL12 receptor CXCR4 occludes most of hNGFp effects. These findings have significant therapeutic implications: (i) we established that a widespread exposure of the brain is required for nerve growth factor to fully exert its neuroprotective actions; and (ii) we have identified a new anti-neurodegenerative pathway as a broad target for new therapeutic opportunities for neurodegenerative diseases.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Córtex Cerebral/metabolismo , Quimiocina CXCL12/metabolismo , Transtornos da Memória/tratamento farmacológico , Fator de Crescimento Neural/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Dor/induzido quimicamente , Placa Amiloide/tratamento farmacológico , Administração Intranasal , Animais , Comportamento Animal , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural/administração & dosagem , Fator de Crescimento Neural/efeitos adversos , Receptores CXCR4/antagonistas & inibidoresRESUMO
Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria-ER contact sites (MERCS) are altered in Alzheimer's disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of Aß as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that Aß increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to Aß. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER-mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that Aß can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Animais , Autofagossomos/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Retículo Endoplasmático/fisiologia , Feminino , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Regulação para Cima/fisiologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Alterations of adult neurogenesis have been reported in several Alzheimer's disease (AD) animal models and human brains, while defects in this process at presymptomatic/early stages of AD have not been explored yet. To address this, we investigated potential neurogenesis defects in Tg2576 transgenic mice at 1.5 months of age, a prodromal asymptomatic age in terms of Aß accumulation and neurodegeneration. We observe that Tg2576 resident and SVZ-derived adult neural stem cells (aNSCs) proliferate significantly less. Further, they fail to terminally differentiate into mature neurons due to pathological, tau-mediated, and microtubule hyperstabilization. Olfactory bulb neurogenesis is also strongly reduced, confirming the neurogenic defect in vivo. We find that this phenotype depends on the formation and accumulation of intracellular A-beta oligomers (AßOs) in aNSCs. Indeed, impaired neurogenesis of Tg2576 progenitors is remarkably rescued both in vitro and in vivo by the expression of a conformation-specific anti-AßOs intrabody (scFvA13-KDEL), which selectively interferes with the intracellular generation of AßOs in the endoplasmic reticulum (ER). Altogether, our results demonstrate that SVZ neurogenesis is impaired already at a presymptomatic stage of AD and is caused by endogenously generated intracellular AßOs in the ER of aNSCs. From a translational point of view, impaired SVZ neurogenesis may represent a novel biomarker for AD early diagnosis, in association to other biomarkers. Further, this study validates intracellular Aß oligomers as a promising therapeutic target and prospects anti-AßOs scFvA13-KDEL intrabody as an effective tool for AD treatment.
Assuntos
Doença de Alzheimer/complicações , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Espaço Intracelular/metabolismo , Degeneração Neural/complicações , Neurogênese , Multimerização Proteica , Animais , Diferenciação Celular , Proliferação de Células , Humanos , Camundongos Transgênicos , Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/patologia , Bulbo Olfatório/metabolismo , Conformação ProteicaRESUMO
GABAergic transmission regulates neuronal excitability, dendritic integration of synaptic signals and oscillatory activity, thought to be involved in high cognitive functions. By anchoring synaptic receptors just opposite to release sites, the scaffold protein gephyrin plays a key role in these tasks. In addition, by regulating GABAA receptor trafficking, gephyrin contributes to maintain, at the network level, an appropriate balance between Excitation (E) and Inhibition (I), crucial for information processing. An E/I imbalance leads to neuropsychiatric disorders such as epilepsy, schizophrenia and autism. In this article, we exploit a previously published computational method to fit spontaneous synaptic events, using a simplified model of the subcellular pathways involving gephyrin at inhibitory synapses. The model was used to analyze experimental data recorded under different conditions, with the main goal to gain insights on the possible consequences of gephyrin block on IPSCs. The same approach can be useful, in general, to analyze experiments designed to block a single protein. The results suggested possible ways to correlate the changes observed in the amplitude and time course of individual events recorded after different experimental protocols with the changes that may occur in the main subcellular pathways involved in gephyrin-dependent transsynaptic signaling.
RESUMO
BACKGROUND: A consistent proportion of individuals at risk for Alzheimer's disease show intact cognition regardless of the extensive accumulation of amyloid-ß (Aß) peptide in their brain. Several pieces of evidence indicate that overactivation of brain regions negative for Aß can compensate for the underactivation of Aß-positive ones to preserve cognition, but the underlying synaptic changes are still unexplored. METHODS: Using Golgi staining, we investigate how dendritic spines rearrange following contextual fear conditioning (CFC) in the hippocampus and amygdala of presymptomatic Tg2576 mice, a genetic model for Aß accumulation. A molecular biology approach combined with intrahippocampal injection of a γ-secretase inhibitor evaluates the impact of Aß fluctuations on spine rearrangements. RESULTS: Encoding of CFC increases Aß oligomerization in the hippocampus but not in the amygdala of Tg2576 mice. The presence of Aß oligomers predicts vulnerability to network dysfunctions, as low c-Fos activation and spine maturation are detected in the hippocampus of Tg2576 mice upon recall of CFC memory. Rather, enhanced c-Fos activation and new spines are evident in the amygdala of Tg2576 mice compared with wild-type control mice. Preventing Aß increase in the hippocampus of Tg2576 mice restores CFC-associated spine changes to wild-type levels in both the hippocampus and amygdala. CONCLUSIONS: Our study provides the first evidence of neural compensation consisting of enhanced synaptic activity in brain regions spared by Aß load. Furthermore, it unravels an activity-mediated feedback loop through which neuronal activation during CFC encoding favors Aß oligomerization in the hippocampus and prevents synaptic rearrangements in this region.
Assuntos
Doença de Alzheimer/fisiopatologia , Encéfalo/fisiopatologia , Espinhas Dendríticas/fisiologia , Medo/fisiologia , Memória , Vias Neurais/fisiopatologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade NeuronalRESUMO
Nerve growth factor (NGF) exerts a trophic, antiapoptotic action on several neuronal targets, including the clonal cell line PC12. In the current study, we demonstrate that withdrawal of this neurotrophin from PC12 differentiated cells causes overproduction of amyloid-beta (Abeta) peptides, which are the most toxic protein fragments directly implicated in the development of Alzheimer disease (AD), concomitantly with cell death by apoptosis. Abeta production and apoptotic death, occurring after withdrawal from NGF-differentiated PC12 cells, are completely inhibited by beta- and gamma-secretase inhibitors and by antibodies directed against Abeta peptides, favouring maintenance of PC12 morphology and neuritic network. These peptides are partially released and largely deposited as aggregates only soluble with strong detergent treatment generally employed to dissolve senile plaques. Furthermore, partial silencing of APP mRNA, by siRNA, reduces not only the extent of Abeta production but also apoptotic death. Abeta production and apoptosis are also induced in differentiated PC12 cells by kinase inhibitors of Trk-A, the high affinity receptor of NGF and, in this case, the co-incubation with beta- and gamma-secretase inhibitors totally revert apoptosis.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Apoptose/fisiologia , Fatores de Crescimento Neural/genética , Células PC12/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Anticorpos Monoclonais , Western Blotting , Células Cultivadas , Tratamento Farmacológico , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Inativação Gênica , RNA Mensageiro/genética , RatosRESUMO
Chronic anemia is common in liver cirrhosis. In this setting, the pathogenesis of anemia is complex and multifactorial. Spur cell anemia is a serious disorder in cirrhotic patients and is associated with poor prognosis. Liver transplantation constitutes the only therapeutic tool. We report a case with severe spur cell anemia in alcoholic liver cirrhosis. In the attempt to investigate the origin of the disorder, we have evaluated the lipoprotein profile and found a significant reduction of apolipoprotein AI and HDL3 subclass as a possible cause of the disease.
RESUMO
The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.
Assuntos
Região Variável de Imunoglobulina/imunologia , Biblioteca de Peptídeos , Proteômica/métodos , Vetores Genéticos , Genômica/métodos , PlasmídeosRESUMO
Aß oligomers (AßOs) are crucially involved in Alzheimer's Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aß assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AßOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AßOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aß precursor protein. The anti-AßOs intrabody selectively intercepts critical AßO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aß undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AßOs as key targets for AD treatment and presents CSI as a potential targeting strategy.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Retículo Endoplasmático/metabolismo , Espaço Intracelular/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Western Blotting , Células CHO , Cricetinae , Cricetulus , Espaço Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Anticorpos de Cadeia Única/metabolismo , Frações Subcelulares/metabolismoRESUMO
The 7WD4 and 7PA2 cell lines, widely used as cellular models for Alzheimer's disease (AD), have been used to investigate the effects of amyloid-ß protein precursor overexpression and amyloid-ß (Aß) oligomer accumulation on mitochondrial function. Under standard culture conditions, both cell lines, compared to Chinese hamster ovary (CHO) control cells, displayed an ~5% decrease of O2 respiration as sustained by endogenous substrates. Functional impairment of the respiratory chain was found distributed among the protein complexes, though more evident at the level of complex I and complex IV. Measurements of ATP showed that its synthesis by oxidative phosphorylation is decreased in 7WD4 and 7PA2 cells by ~25%, this loss being partly compensated by glycolysis (Warburg effect). Compensation proved to be more efficient in 7WD4 than in 7PA2 cells, the latter cell line displaying the highest reactive oxygen species production. The strongest deficit was observed in mitochondrial membrane potential that is almost 40% and 60% lower in 7WD4 and 7PA2 cells, respectively, in comparison to CHO controls. All functional parameters point to a severe bioenergetic impairment of the AD cells, with the extent of mitochondrial dysfunction being correlated to the accumulation of Aß peptides and oligomers.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/fisiologia , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Mitocôndrias/patologiaRESUMO
Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( nâ=â8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.
Assuntos
Amiloide/metabolismo , Progressão da Doença , Transtornos da Memória/prevenção & controle , Fator de Crescimento Neural/administração & dosagem , Fator de Crescimento Neural/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Nociceptividade/efeitos dos fármacos , Administração Intranasal , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutação , Fator de Crescimento Neural/efeitos adversos , Fator de Crescimento Neural/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/farmacologia , Fosfolipase C gama/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinaptofisina/metabolismoRESUMO
Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimer's disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.
Assuntos
Doença de Alzheimer/metabolismo , Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular Tumoral , Feminino , Hipocampo/citologia , Humanos , Mitocôndrias/patologia , Degeneração Neural/patologia , Neuroblastoma , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Gravidez , Ratos , Ratos Wistar , Solubilidade , Sinapses/patologiaRESUMO
The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Abeta) peptide is of great interest for research into Alzheimer's disease (AD). We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Abeta single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Abeta. A LexA-Abeta1-42 fusion protein was expressed in yeast cells, as the "intracellular antigen". Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Abeta-immunized mice. This led to the isolation of 18 different anti-Abeta scFvs, which bind Abeta both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Abeta scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Abeta oligomers with respect to monomeric Abeta, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Abeta1-42; in particular, the scFvs selected from Abeta-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Abeta scFvs were shown to recognize in vivo-produced Abeta "deposits" in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Abeta oligomer-induced toxicity and synaptic binding of Abeta oligomers in neuronal cultured cells. The properties of these anti-Abeta antibody domains, as well as their direct availability for intra- or extra-cellular "genetic delivery" make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Abeta oligomers.