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While Parkinson's disease remains clinically defined by cardinal motor symptoms resulting from nigrostriatal degeneration, it is now appreciated that the disease commonly consists of multiple pathologies, but it is unclear where these co-pathologies occur early in disease and whether they are responsible for the nigrostriatal degeneration. For the past number of years, we have been studying a well-characterized cohort of subjects with motor impairment that we have termed mild motor deficits. Motor deficits were determined on a modified and validated Unified Parkinson's Disease Rating Scale III but were insufficient in degree to diagnose Parkinson's disease. However, in our past studies, cases in this cohort had a selection bias, as both a clinical syndrome in between no motor deficits and Parkinson's disease, plus nigral Lewy pathology as defined post-mortem, were required for inclusion. Therefore, in the current study, we only based inclusion on the presence of a clinical phenotype with mild motor impairment insufficient to diagnose Parkinson's disease. Then, we divided this group further based upon whether or not subjects had a synucleinopathy in the nigrostriatal system. Here we demonstrate that loss of nigral dopaminergic neurons, loss of putamenal dopaminergic innervation and loss of the tyrosine hydroxylase-phenotype in the substantia nigra and putamen occur equally in mild motor deficit groups with and without nigral alpha-synuclein aggregates. Indeed, the common feature of these two groups is that both have similar degrees of AT8 positive phosphorylated tau, a pathology not seen in the nigrostriatal system of age-matched controls. These findings were confirmed with early (tau Ser208 phosphorylation) and late (tau Ser396/Ser404 phosphorylation) tau markers. This suggests that the initiation of nigrostriatal dopaminergic neurodegeneration occurs independently of alpha-synuclein aggregation and can be tau mediated.
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Doença de Parkinson , Transtornos Parkinsonianos , Sinucleinopatias , Humanos , Doença de Parkinson/complicações , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , Transtornos Parkinsonianos/patologia , Sinucleinopatias/patologia , Putamen/metabolismo , Substância Negra/metabolismo , DopaminaRESUMO
The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.
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Encéfalo/metabolismo , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Sinucleinopatias/metabolismo , Globinas beta/metabolismoRESUMO
INTRODUCTION: The understanding of the pathological events in Alzheimer's disease (AD) has advanced dramatically, but the successful translation from rodent models into efficient human therapies is still problematic. METHODS: To examine how tau pathology can develop in the primate brain, we injected 12 macaques with a dual tau mutation (P301L/S320F) into the entorhinal cortex (ERC). An investigation was performed using high-resolution microscopy, magnetic resonance imaging (MRI), positron emission tomography (PET), and fluid biomarkers to determine the temporal progression of the pathology 3 and 6 months after the injection. RESULTS: Using quantitative microscopy targeting markers for neurodegeneration and neuroinflammation, as well as fluid and imaging biomarkers, we detailed the progression of misfolded tau spreading and the consequential inflammatory response induced by glial cells. DISCUSSION: By combining the analysis of several in vivo biomarkers with extensive brain microscopy analysis, we described the initial steps of misfolded tau spreading and neuroinflammation in a monkey model highly translatable to AD patients. HIGHLIGHTS: Dual tau mutation delivery in the entorhinal cortex induces progressive tau pathology in rhesus macaques. Exogenous human 4R-tau coaptates monkey 3R-tau during transneuronal spread, in a prion-like manner. Neuroinflammatory response is coordinated by microglia and astrocytes in response to tau pathology, with microglia targeting early tau pathology, while astrocytes engaged later in the progression, coincident with neuronal death. Monthly collection of CSF and plasma revealed a profile of changes in several AD core biomarkers, reflective of neurodegeneration and neuroinflammation as early as 1 month after injection.
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BACKGROUND: Neurturin is a member of the glial cell line-derived neurotrophic factor family of neurotrophic factors and has the potential to protectdegenerating dopaminergic neurons. OBJECTIVE: Here, we performed post-mortem studies on two patients with advanced Parkinson's disease that survived 10 years following AAV-neurturin gene (Cere120) delivery to verify long-term effects of trophic factor neurturin. METHODS: Cere120 was delivered to the putamen bilaterally in one case and to the putamen plus substantia nigra bilaterally in the second. Immunohistochemistry was used to examine neurturin, Rearranged during transfection(RET), phosphor-S6, and tyrosine hydroxylase expressions, inflammatory reactions, and α-synuclein accumulation. RESULTS: In both patients there was persistent, albeit limited, neurturin expression in the putamen covering 1.31% to 5.92% of the putamen. Dense staining of tyrosine hydroxylase-positive fibers was observed in areas that contained detectable neurturin expression. In substantia nigra, neurturin expression was detected in 11% of remaining melanin-containing neurons in the patient with combined putamenal and nigral gene delivery, but not in the patient with putamenal gene delivery alone. Tyrosine hydroxylase positive neurons were 66% to 84% of remaining neuromelanin neurons in substantia nigra with Cere120 delivery and 23% to 24% in substantia nigra without gene delivery. More RET and phosphor-S6 positive neurons were observed in substantia nigra following nigral Cere120. Inflammatory and Lewy pathologies were similar in substantia nigra with or without Cere120 delivery. CONCLUSIONS: This study provides evidence of long-term persistent transgene expression and bioactivity following gene delivery to the nigrostriatal system. Therefore, future efforts using gene therapy for neurodegenerative diseases should consider means to enhance remaining dopamine neuron function and stop pathological propagation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Doença de Parkinson , Humanos , Doença de Parkinson/genética , Doença de Parkinson/terapia , Doença de Parkinson/metabolismo , Neurturina/genética , Neurturina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Neurônios/metabolismo , Terapia Genética , Substância Negra/metabolismoRESUMO
The failure of glial cell derived neurotropic factor to be efficacious in blinded clinical trials for Parkinson's disease may be due to alterations in signaling receptors and downstream signaling molecules. To test this hypothesis, brain sections were obtained from older adults with no motor deficit (n = 6), minimal motor deficits (n = 10), and clinical diagnosis of Parkinson's disease (n = 10) who underwent motor examination proximate to death. Quantitative unbiased stereology and densitometry were performed to analyze RET and phosphorylated ribosomal protein S6 expression in nigral neurons. Individuals with no motor deficit had extensive and intense RET and phosphorylated ribosomal protein S6 immunoreactive neurons in substantia nigra. The number and staining intensity of RET-immunoreactive neurons were reduced moderately in subjects with minimal motor deficits and severely reduced in Parkinson's disease relative to no motor deficit group. The number and staining intensity of phosphorylated ribosomal protein S6 was more markedly reduced in both subjects with minimal motor deficits and Parkinson's disease. Reductions in levels of RET and phosphorylated ribosomal protein S6 were recapitulated in a non-human primate genetic Parkinson's disease model based on over-expression of human mutant α-synuclein (A53T). These data indicate that for neurotrophic factors to be effective in patients with minimal motor deficits or PD, these factors would likely have to upregulate RET and phosphorylated ribosomal protein S6 immunoreactive neurons in substantia nigra .
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Encéfalo/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Neurturina/metabolismo , Doença de Parkinson/metabolismo , Sintomas Prodrômicos , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteína S6 Ribossômica/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Densitometria , Feminino , Humanos , Macaca fascicularis , Masculino , Doença de Parkinson/fisiopatologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Transdução de Sinais , alfa-Sinucleína/genéticaRESUMO
We performed post-mortem studies on two patients with advanced Parkinson's disease 8 and10 years following AAV2-neurturin (CERE120) gene therapy, the longest post-mortem trophic factor gene therapy cases reported to date. CERE120 was delivered to the putamen bilaterally in one case (10 years post-surgery), and to the putamen plus the substantia nigra bilaterally in the second (8 years post-surgery). In both patients there was persistent, albeit limited, neurturin expression in the putamen covering â¼3-12% of the putamen. In the putamen, dense staining of tyrosine hydroxylase-positive fibres was observed in areas that contained detectable neurturin expression. In the substantia nigra, neurturin expression was detected in 9.8-18.95% and 22.02-39% of remaining melanin-containing neurons in the patient with putamenal and combined putamenal and nigral gene delivery, respectively. Melanized neurons displayed intense tyrosine hydroxylase and RET proto-oncogene expression in nigral neurons in the patient where CERE120 was directly delivered to the nigra. There was no difference in the degree of Lewy pathology in comparison to untreated control patients with Parkinson's disease, and α-synuclein aggregates were detected in neurons that also stained for neurturin, RET, and tyrosine hydroxylase. These changes were not associated with antiparkinsonian benefits likely due to the limited neurturin expression. This study provides the longest term evidence of persistent transgene expression following gene delivery to the CNS and the first human results when targeting both the terminal fields in the putamen as well as the originating nigral neurons.
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Terapia Genética , Neurturina/biossíntese , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Humanos , Corpos de Lewy/metabolismo , Melaninas/imunologia , Pessoa de Meia-Idade , Neurônios/imunologia , Neurturina/administração & dosagem , Doença de Parkinson/imunologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret/biossíntese , Putamen/imunologia , Putamen/metabolismo , Substância Negra/imunologia , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/imunologia , alfa-Sinucleína/metabolismoRESUMO
We observed Lewy pathology in healthy embryonic dopamine neurons implanted into the striatum of patients with advanced Parkinson's disease. In the present study we examined the temporal relationship between the presence of inflammation with activated microglia and the emergence of α-synuclein pathology. Inflammation with activated microglia was observed in all grafts and at all time points examined between 18 months and 16 years as determined by both CD45 and TMEM119 staining. In contrast, α-synuclein was not detected at 18 months, only diffuse monomeric α-synuclein staining was observed at 4 years, and α-synuclein aggregates were not observed until 14-16 years after transplantation. Thus, there is evidence of inflammation and microglial activation in graft deposits long before the accumulation of α-synuclein pathology in implanted dopamine neurons. These observations raise the possibility that microglial activation contributes to the development of α-synuclein pathology, and supports the concept that microglia play an integral role in the propagation and spread of α-synuclein pathology.
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Neurônios Dopaminérgicos/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Animais , Neurônios Dopaminérgicos/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Ativação de Macrófagos/fisiologia , Camundongos Transgênicos , Microglia/patologia , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismoRESUMO
Several studies have demonstrated that intrastriatal injections of fibrillar α-synuclein in rodent brain induced a Parkinson's disease-like propagation of Lewy body pathology with significant nigrostriatal neurodegeneration. This study evaluated the pathological features when exogenous α-synuclein preformed fibrils were injected into the putamen of non-human primates. Eight cynomolgus monkeys received unilateral intraputamen injections of α-synuclein preformed fibrils and four monkeys received sham surgery. Monkeys were assessed with 123I-PE2I single-photon emission computerized tomography scans targeting the dopamine transprter at baseline, 3, 6, 9, 12, and 15 months. Imaging revealed a robust increase in dopamine transporter binding, an effect confirmed by port-mortem immunohistochemical analyses, suggesting that upregulation of dopamine transporter occurs as part of an early pathological process. Histochemistry and immunohistochemistry revealed that α-synuclein preformed fibrils injections into the putamen induced intraneuronal inclusions positive for phosphorylated α-synuclein in ipsilateral substantia nigra and adjacent to the injection site. α-Synuclein inclusions were thioflavin-S-positive suggesting that the inclusions induced by α-synuclein preformed fibrils exhibited pathological properties similar to amyloid-like Lewy body pathology in Parkinson's disease brains. The α-synuclein preformed fibrils resulted in Lewy pathology in the ipsilateral substantia nigra with significant reduction (-29.30%) of dopaminergic neurons as compared with controls. Nigral neurons with α-synuclein inclusions exhibited a phenotypic downregulation of the dopamine markers tyrosine hydroxylase and Nurr1. Taken together, our findings demonstrate that α-synuclein preformed fibrils induce a synucleinopathy in non-human primates with authentic Lewy pathology and nigrostriatal changes indicative of early Parkinson's disease.
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Neostriado/metabolismo , Neostriado/patologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , Animais , Contagem de Células , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Imuno-Histoquímica , Corpos de Lewy/patologia , Macaca fascicularis , Microinjeções , Neostriado/diagnóstico por imagem , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Putamen , Substância Negra/metabolismo , Substância Negra/patologia , Sinucleinopatias/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/administração & dosagemRESUMO
OBJECTIVE: Understanding the pathological changes underlying mild motor features of the eldery and defining a patient population with prodromal Parkinson disease (PD) are of great clinical importance. It remains unclear, however, how to accurately and specifically diagnose prodromal PD. We examined whether older adults with minimal parkinsonian motor features have nigrostriatal degeneration and α-synuclein pathology consistent with prodromal PD. METHODS: Brain sections were obtained from older adults with a clinical diagnosis of PD (n = 21) and without a clinical diagnosis of PD (n = 27) who underwent motor examination proximate to death. Cases without PD were further dichotomized into no motor deficit (n = 9) or minimal motor features (n = 18) groups using a modified Unified Parkinson's Disease Rating Scale. We performed quantitative unbiased stereological analyses of dopaminergic neurons/terminals and α-synuclein accumulation in the nigrostriatal system. RESULTS: In all subjects with minimal motor features, there were significant reductions in dopaminergic neurons and terminals in the substantia nigra and putamen that were intermediate between subjects with no motor deficit and PD. Phosphorylated α-synuclein inclusions were observed in the substantia nigra that were of similar density to what was seen in PD. Furthermore, there was greater Lewy neuritic pathology in the putamen relative to PD patients. Lastly, neurons with α-synuclein inclusions displayed reductions in tyrosine hydroxylase expression that were comparable in subjects with both minimal motor features and PD. INTERPRETATION: Minimal motor features in older adults may represent prodromal PD and identify at-risk individuals for testing putative neuroprotective interventions that could slow or prevent PD progression. Ann Neurol 2018;83:562-574.
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Corpo Estriado/patologia , Transtornos das Habilidades Motoras/patologia , Doença de Parkinson/patologia , Sintomas Prodrômicos , Substância Negra/patologia , Idoso , Idoso de 80 Anos ou mais , Corpo Estriado/metabolismo , Feminino , Humanos , Masculino , Transtornos das Habilidades Motoras/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Methods: Human ARPE-19 cells engineered to secrete high levels of the glial cell line-derived neurotrophic factor (GDNF) were encapsulated into hollow fiber membranes. The devices were implanted into the rat striatum 1 week prior to striatal quinolinic acid injections. Animals were evaluated using a battery of validated motor tests, and histology was performed to determine the extent of GDNF diffusion and associated prevention of neuronal cell loss and behavioral deficits. Results: Encapsulated cell-based delivery of GDNF produced widespread distribution of GDNF throughout the entire implanted striatum. Stereological estimates of striatal neuron number and volume of lesion size revealed that GDNF delivery resulted in near complete neuroprotection. Conclusions: Delivery of neurotrophic molecules such as GDNF using encapsulated cells has reached a technological point where clinical evaluation is justified. Because GDNF has been effective in animal models of Parkinson's disease, stroke, epilepsy, and Huntington's disease, among other debilitating neurodegenerative diseases, encapsulated cell-based delivery of GDNF might represent one innovative means of slowing the neural degeneration seen in a myriad of currently untreatable neurological diseases.
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Corpo Estriado/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Ácido Quinolínico/toxicidade , Animais , Encapsulamento de Células , Linhagem Celular , Sistemas de Liberação de Medicamentos , Humanos , Células LLC-PK1 , Masculino , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley , SuínosRESUMO
OBJECTIVE: The main goal of dopamine cell replacement therapy in Parkinson disease (PD) is to provide clinical benefit mediated by graft survival with nigrostriatal reinnervation. We report a dichotomy between graft structure and clinical function in a patient dying 16 years following fetal nigral grafting. METHODS: A 55-year-old levodopa-responsive woman with PD received bilateral putaminal fetal mesencephalic grafts as part of an NIH-sponsored double-blind sham-controlled trial. The patient never experienced clinical benefit, and her course was complicated by the development of graft-related dyskinesias. Fluorodopa positron emission tomography demonstrated significant increases postgrafting bilaterally. She experienced worsening of parkinsonism with severe dyskinesias, and underwent subthalamic nucleus deep brain stimulation 8 years after grafting. She died 16 years after transplantation. RESULTS: Postmortem analyses confirmed the diagnosis of PD and demonstrated >300,000 tyrosine hydroxylase (TH)-positive grafted cells per side with normalized striatal TH-immunoreactive fiber innervation and bidirectional synaptic connectivity. Twenty-seven percent and 17% of grafted neurons were serine 129-phosphorylated α-synuclein positive in the left and right putamen, respectively. INTERPRETATION: These findings represent the largest number of surviving dopamine neurons and the densest and most widespread graft-mediated striatal dopamine reinnervation following a transplant procedure reported to date. Despite this, clinical recovery was not observed. Furthermore, the grafts were associated with a form of dyskinesias that resembled diphasic dyskinesia and persisted in the off-medication state. We hypothesize that the grafted cells produced a low level of dopamine sufficient to cause a levodopa-independent continuous form of diphasic dyskinesias, but insufficient to provide an antiparkinsonian benefit. ANN NEUROL 2017;81:46-57.
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Neurônios Dopaminérgicos/metabolismo , Sobrevivência de Enxerto , Mesencéfalo/transplante , Doença de Parkinson/cirurgia , Transplante de Tecido Encefálico , Neurônios Dopaminérgicos/ultraestrutura , Feminino , Humanos , Pessoa de Meia-Idade , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.
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Axônios/patologia , Encéfalo/patologia , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Idoso , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Expressão Gênica , Genes Reporter , Humanos , Doença de Huntington/diagnóstico , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Pessoa de Meia-Idade , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregação Patológica de Proteínas , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Índice de Gravidade de DoençaRESUMO
Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.
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Proteínas Amiloidogênicas/metabolismo , Transporte Biológico/fisiologia , Vesículas Transportadoras/metabolismo , Animais , Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Feminino , Fluoresceínas , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Masculino , Neurônios/metabolismo , Neurônios/ultraestrutura , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosfatidilgliceróis , Ratos , Vesículas Transportadoras/ultraestrutura , Lipossomas Unilamelares , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: This study aimed to assess the effect of dehydroepiandrosterone (DHEA) plus climen (estradiol valerate and cyproterone acetate drug combination) on infertility patients with diminished ovarian reserve (DOR) and to determine if the combination of DHEA plus climen is superior to DHEA alone in improving ovarian response. METHODS: A total of 124 women were randomized into the DHEA group (n = 64) and the DHEA plus climen group (n = 60) for 12 weeks before being subjected to in-vitro fertilization (IVF) cycles. To investigate if there is a FSH-related difference on the effect of the addition of climen, the DHEA group and the DHEA plus climen group were further divided into four subgroups according to a basal FSH level cut-off of 10 mIU/ml. We performed a comparison of Day 3 blood samples before and after treatment and IVF outcome parameters, including AMH, FSH, E2, AFC, oocytes retrieved, MII oocyte numbers, embryo numbers and accumulated embryo scores. RESULTS: After 12 weeks of pretreatment, the DHEA plus climen group demonstrated a significantly higher level of AMH (P = 0.001) and a significantly lower level of FSH (P = 0.001) compared with the DHEA group. When the two groups were divided into four subgroups based on the FSH cut-off of 10 mIU/mL, a significant increase of AMH (P = 0.034) was found in the high-FSH DHEA plus climen group, whereas there was no significant difference in the high-FSH DHEA group (P = 0.322). A significantly higher accumulated score of embryos was observed in the low-FSH DHEA plus climen group compared with the low-FSH DHEA group (P = 0.034). CONCLUSIONS: These observations suggest that patients with DOR of a low-FSH level might benefit more from DHEA plus climen supplementation than from DHEA supplementation alone.
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Acetato de Ciproterona/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Estradiol/análogos & derivados , Reserva Ovariana , Indução da Ovulação/métodos , Adulto , Hormônio Antimülleriano/sangue , Acetato de Ciproterona/administração & dosagem , Desidroepiandrosterona/administração & dosagem , Combinação de Medicamentos , Quimioterapia Combinada , Estradiol/administração & dosagem , Estradiol/uso terapêutico , Feminino , Fertilização in vitro , Hormônio Foliculoestimulante/sangue , Humanos , Infertilidade Feminina/tratamento farmacológico , Gravidez , Taxa de Gravidez , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND/AIMS: The lack of available beta cells greatly limits the use of beta cell transplantation as a therapy for diabetes. Thus, generation of beta cells from other sources is substantially required. Pax4 has been shown to induce reprograming of alpha cells into beta cells during embryogenesis. Nevertheless, whether expression of Pax4 in adult alpha cells could trigger this alpha-to-beta cell reprogramming is unknown. METHODS: Here we generated an adeno-associated virus carrying Pax4 and GFP under a CMV promoter (AAV-Pax4). We used AAV-Pax4 to transduce a mouse alpha cell line in vitro, and to transduce primary alpha cells in diabetic mice. Reprogramming was examined by double immunostaining and by changes in beta cell number. The effects on blood glucose were evaluated by fasting blood glucose and glucose response. RESULTS: In vitro, Pax4 overexpression neither induced insulin expression, nor suppressed glucagon expression in alpha cells. In vivo, Pax4 overexpression failed to increase beta cell number, and did not alter hyperglycemia and glucose response in diabetic mice. CONCLUSION: Pax4 expression is not sufficient to transduce pancreatic alpha cells into beta cells. Overexpression of Pax4 in alpha cells may not increase functional beta cell number in diabetic patients.
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Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Pâncreas/citologia , Animais , Glicemia/metabolismo , Linhagem Celular , Dependovirus/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Camundongos , Fatores de Transcrição Box Pareados/genética , Pâncreas/metabolismoRESUMO
The discovery of alpha-synuclein's prion-like behaviors in mammals, as well as a non-Mendelian type of inheritance, has led to a new concept in biology, the "prion hypothesis" of Parkinson's disease. The misfolding and aggregation of alpha-synuclein (α-syn) within the nervous system occur in many neurodegenerative diseases including Parkinson's disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). The molecular basis of synucleinopathies appears to be tightly coupled to α-syn's conformational conversion and fibril formation. The pathological form of α-syn consists of oligomers and fibrils with rich in ß-sheets. The conversion of its α-helical structure to the ß-sheet rich fibril is a defining pathologic feature of α-syn. These kinds of disorders have been classified as protein misfolding diseases or proteopathies which share key biophysical and biochemical characteristics with prion diseases. In this review, we highlight α-syn's prion-like activities in PD and PD models, describe the idea of a prion-like mechanism contributing to PD pathology, and discuss several key molecules that can modulate the α-syn accumulation and propagation.
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Doença de Parkinson/metabolismo , Príons/metabolismo , Animais , Humanos , Camundongos , alfa-Sinucleína/metabolismoRESUMO
Both the misfolding of α-synuclein and mitochondrial dysfunction are considered two major contributors to Parkinson's disease (PD). However, the relationship between the two in normal and PD states remains unclear. Here, we report that voltage-dependent anion channel 1 (VDAC1), a major component of the outer mitochondrial membrane known to regulate mitochondrial functions, is down-regulated in response to α-synuclein accumulation and aggregation. Stereological analysis revealed that 58.33% of the neurons were VDAC1 immunoreactive in the remaining neuromelanin laden neurons in the PD group while 87.48% of the nigral neurons were VDAC1 immunoreactive in the age-matched control group. The relative levels of VDAC1 were significantly decreased in PD nigral neurons when compared to age-matched controls. In PD, this decrease was significantly greater in nigral neurons with α-synuclein inclusions. VDAC1 was observed in fibers with granular α-synuclein but not in fibers with aggregated α-synuclein. Viral vector-mediated overexpression of mutant human α-synuclein (A30P) in rats resulted in significantly decreased VDAC1 in nigral neurons and striatal fibers. These results indicate that mitochondrial function associated with VDAC1 is decreased in sporadic and experimental PD, and this decrease is associated with α-synuclein accumulation and aggregation.
Assuntos
Encéfalo/metabolismo , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/patologia , Transtornos Parkinsonianos/patologia , Ratos Sprague-Dawley , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/genéticaRESUMO
The pace of nigrostriatal degeneration, both with regards to striatal denervation and loss of melanin and tyrosine hydroxylase-positive neurons, is poorly understood especially early in the Parkinson's disease process. This study investigated the extent of nigrostriatal degeneration in patients with Parkinson's disease at different disease durations from time of diagnosis. Brains of patients with Parkinson's disease (n=28) with post-diagnostic intervals of 1-27 years and normal elderly control subjects (n=9) were examined. Sections of the post-commissural putamen and substantia nigra pars compacta were processed for tyrosine hydroxylase and dopamine transporter immunohistochemistry. The post-commissural putamen was selected due to tissue availability and the fact that dopamine loss in this region is associated with motor disability in Parkinson's disease. Quantitative assessments of putaminal dopaminergic fibre density and stereological estimates of the number of melanin-containing and tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta (both in total and in subregions) were performed by blinded investigators in cases where suitable material was available (n=17). Dopaminergic markers in the dorsal putamen showed a modest loss at 1 year after diagnosis in the single case available for study. There was variable (moderate to marked) loss, at 3 years. At 4 years post-diagnosis and thereafter, there was virtually complete loss of staining in the dorsal putamen with only an occasional abnormal dopaminergic fibre detected. In the substantia nigra pars compacta, there was a 50-90% loss of tyrosine hydroxylase-positive neurons from the earliest time points studied with only marginal additional loss thereafter. There was only a â¼10% loss of melanized neurons in the one case evaluated 1 year post-diagnosis, and variable (30 to 60%) loss during the first several years post-diagnosis with more gradual and subtle loss in the second decade. At all time points, there were more melanin-containing than tyrosine hydroxylase-positive cells. Loss of dopaminergic markers in the dorsal putamen occurs rapidly and is virtually complete by 4 years post-diagnosis. Loss of melanized nigral neurons lags behind the loss of dopamine markers. These findings have important implications for understanding the nature of Parkinson's disease neurodegeneration and for studies of putative neuroprotective/restorative therapies.
Assuntos
Corpo Estriado/patologia , Neurônios/patologia , Doença de Parkinson/patologia , Substância Negra/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
BACKGROUND: Nerve growth factor (NGF) is an endogenous neurotrophic-factor protein with the potential to restore function and to protect degenerating cholinergic neurons in Alzheimer's disease (AD), but safe and effective delivery has proved unsuccessful. METHODS: Gene transfer, combined with stereotactic surgery, offers a potential means to solve the long-standing delivery obstacles. An open-label clinical trial evaluated the safety and tolerability, and initial efficacy of three ascending doses of the genetically engineered gene-therapy vector adeno-associated virus serotype 2 delivering NGF (AAV2-NGF [CERE-110]). Ten subjects with AD received bilateral AAV2-NGF stereotactically into the nucleus basalis of Meynert. RESULTS: AAV2-NGF was safe and well-tolerated for 2 years. Positron emission tomographic imaging and neuropsychological testing showed no evidence of accelerated decline. Brain autopsy tissue confirmed long-term, targeted, gene-mediated NGF expression and bioactivity. CONCLUSIONS: This trial provides important evidence that bilateral stereotactic administration of AAV2-NGF to the nucleus basalis of Meynert is feasible, well-tolerated, and able to produce long-term, biologically active NGF expression, supporting the initiation of an ongoing multicenter, double-blind, sham-surgery-controlled trial.
Assuntos
Doença de Alzheimer/terapia , Dependovirus/genética , Terapia Genética/métodos , Fator de Crescimento Neural/genética , Idoso , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Núcleo Basal de Meynert , Estudos de Viabilidade , Feminino , Vetores Genéticos , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Procedimentos Neurocirúrgicos , Tomografia por Emissão de Pósitrons , Técnicas Estereotáxicas , Resultado do TratamentoRESUMO
The progressive loss of the nigrostriatal pathway is a distinguishing feature of Parkinson's disease. As terminal field loss seems to precede cell body loss, we tested whether alterations of axonal transport motor proteins would be early features in Parkinson's disease. There was a decline in axonal transport motor proteins in sporadic Parkinson's disease that preceded other well-known nigral cell-related pathology such as phenotypic downregulation of dopamine. Reductions in conventional kinesin levels precede the alterations in dopaminergic phenotypic markers (tyrosine hydroxylase) in the early stages of Parkinson's disease. This reduction was significantly greater in nigral neurons containing α-synuclein inclusions. Unlike conventional kinesin, reductions in the levels of the cytoplasmic dynein light chain Tctex type 3 subunit were only observed at late Parkinson's disease stages. Reductions in levels of conventional kinesin and cytoplasmic dynein subunits were recapitulated in a rat genetic Parkinson's disease model based on over-expression of human mutant α-synuclein (A30P). Together, our data suggest that α-synuclein aggregation is a key feature associated with reductions of axonal transport motor proteins in Parkinson's disease and support the hypothesis that dopaminergic neurodegeneration following a 'dying-back' pattern involving axonal transport disruption.