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Importance: Parkinson's disease (PD), the second most common neurodegenerative disease, is pathologically characterized by intraneuronal deposition of misfolded alpha-synuclein aggregates (αSyn D ). αSyn D seeding activities in CSF and skin samples have shown great promise in PD diagnosis, but they require invasive procedures. Sensitive and accurate αSyn D seed amplification assay (αSyn-SAA) for more accessible and minimally invasive samples (such as blood and saliva) are urgently needed for PD pathological diagnosis in routine clinical practice. Objective: To develop a sensitive and accurate αSyn-SAA biomarker using blood and saliva samples for sensitive, accurate and minimally invasive PD diagnosis. Design Setting and Participants: This prospective diagnostic study evaluates serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC) without PD at an academic Parkinson's and Movement Disorders Center from February 2020 to March 2024. Patients diagnosed with non-PD parkinsonism were excluded from this analysis. A total of 124 serum samples (82 PD and 42 HC) and 131 saliva samples (83 PD and 48 HC) were collected and examined by αSyn-SAA. Out of the 124 serum donors, a subset of 74 subjects (48 PD and 26 HC) also donated saliva samples during the same visits. PD patients with serum samples had a mean age of 69.21 years (range 44-88); HC subjects with serum samples had a mean age of 66.55 years (range 44-81); PD patients with saliva samples had a mean age of 69.58 years (range 49-87); HC subjects with saliva samples had a mean age of 64.71 years (range 30-81). Main Outcomes and Measures: Serum and/or saliva αSyn D seeding activities from PD and HC subjects were measured by αSyn-SAA using the Real-Time Quaking-Induced Conversion (RT-QuIC) platform. These PD patients had extensive clinical assessments including MDS-UPDRS. For a subset of PD and HC subjects whose serum and saliva samples were both collected during the same visits, the αSyn D seeding activities in both samples from the same subjects were examined, and the diagnostic accuracies for PD based on the seeding activities in either sample alone or both samples together were compared. Results: RT-QuIC analysis of αSyn D seeding activities in the 124 serum samples revealed a sensitivity of 80.49%, a specificity of 90.48%, and an accuracy of 0.9006 (AUC of ROC, 95% CI, 0.8472-0.9539, p <0.0001) for PD diagnosis. RT-QuIC analysis of αSyn D seeding activity in 131 saliva samples revealed a sensitivity of 74.70%, a specificity of 97.92%, and an accuracy of 0.8966 (AUC of ROC, 95% CI, 0.8454-0.9478, p <0.0001). When aSyn D seeding activities in the paired serum-saliva samples from the subset of 48 PD and 26 HC subjects were considered together, sensitivity was 95.83%, specificity was 96.15%, and the accuracy was 0.98 (AUC of ROC, 95% CI, 0.96-1.00, p <0.001), which are significantly better than when αSyn D seeding activities in either serum or saliva were used alone. For the paired serum-saliva samples, when specificity was set at 100% by elevating the αSyn-SAA cutoff values, a sensitivity of 91.7% and an accuracy of 0.9457 were still attained. Detailed correlation analysis revealed that αSyn D seeding activities in the serum of PD patients were correlated inversely with Montreal Cognitive Assessment (MoCA) score ( p =0.04), positively with Hamilton Depression Rating Scale (HAM-D) ( p =0.03), and weakly positively with PDQ-39 cognitive impairment score ( p =0.07). Subgroup analysis revealed that the inverse correlation with MoCA was only seen in males ( p =0.013) and weakly in the ≥70 age group ( p =0.07), and that the positive correlation with HAM-D was only seen in females ( p =0.04) and in the <70 age group ( p =0.01). In contrast, αSyn D seeding activities in the saliva of PD patients were inversely correlated with age at diagnosis ( p =0.02) and the REM sleep behavior disorder (RBD) status ( p =0.04), but subgroup analysis showed that the inverse correlation with age at diagnosis was only seen in males ( p =0.04) and in the <70 age group ( p =0.01). Conclusion and Relevance: Our data show that concurrent RT-QuIC assay of αSyn D seeding activities in both serum and saliva can achieve high diagnostic accuracies comparable to that of CSF αSyn-SAA, suggesting that αSyn D seeding activities in serum and saliva together can potentially be used as a valuable biomarker for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice. αSyn D seeding activities in serum and saliva of PD patients correlate differentially with some clinical characteristics and in an age and sex-dependent manner. KEY POINTS: Question: Are αSyn D seeding activities in serum and saliva together a more sensitive and accurate diagnostic PD biomarker than αSyn D seeding activities in either sample type alone? Are αSyn D seeding activities in either serum or saliva correlated with any clinical characteristics? Findings: Examinations of αSyn D seeding activities in 124 serum samples and 131 saliva samples from PD and heathy control subjects show that αSyn D seeding activities in both serum and saliva samples together can provide significantly more sensitive and accurate diagnosis of PD than either sample type alone. αSyn D seeding activities in serum or saliva exhibit varied inverse or positive correlations with some clinical features in an age and sex-dependent manner. Meaning: αSyn D seeding activities in serum and saliva together can potentially be used as a valuable pathological biomarker for highly sensitive, accurate, and minimally invasive PD diagnosis in routine clinical practice and clinical studies, and αSyn D seeding activities in serum or saliva correlate with some clinical characteristics in an age and sex-dependent manner, suggesting some possible clinical utility of quantitative serum/saliva αSyn-SAA data.
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Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.
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Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Humanos , Priones/genética , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/genética , Síndrome de Creutzfeldt-Jakob/diagnóstico , Síndrome de Creutzfeldt-Jakob/genética , BiomarcadoresRESUMEN
INTRODUCTION: Tumor necrosis factor (TNF) inhibitors are widely used to treat rheumatoid arthritis (RA) and their potential to retard Alzheimer's disease (AD) progression has been reported. However, their long-term effects on the dementia/AD risk remain unknown. METHODS: A propensity scored matched retrospective cohort study was conducted among 40,207 patients with RA within the US Veterans Affairs health-care system from 2000 to 2020. RESULTS: A total of 2510 patients with RA prescribed TNF inhibitors were 1:2 matched to control patients. TNF inhibitor use was associated with reduced dementia risk (hazard ratio [HR]: 0.64, 95% confidence interval [CI]: 0.52-0.80), which was consistent as the study period increased from 5 to 20 years after RA diagnosis. TNF inhibitor use also showed a long-term effect in reducing the risk of AD (HR: 0.57, 95% CI: 0.39-0.83) during the 20 years of follow-up. CONCLUSION: TNF inhibitor use is associated with lower long-term risk of dementia/AD among US veterans with RA.
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Antirreumáticos , Artritis Reumatoide , Demencia , Veteranos , Antirreumáticos/efectos adversos , Artritis Reumatoide/complicaciones , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/epidemiología , Demencia/inducido químicamente , Demencia/epidemiología , Demencia/prevención & control , Humanos , Puntaje de Propensión , Estudios Retrospectivos , Inhibidores del Factor de Necrosis TumoralRESUMEN
Skin α-synuclein deposition is considered a potential biomarker for Parkinson's disease (PD). Real-time quaking-induced conversion (RT-QuIC) is a novel, ultrasensitive, and efficient seeding assay that enables the detection of minute amounts of α-synuclein aggregates. We aimed to determine the diagnostic accuracy, reliability, and reproducibility of α-synuclein RT-QuIC assay of skin biopsy for diagnosing PD and to explore its correlation with clinical markers of PD in a two-center inter-laboratory comparison study. Patients with clinically diagnosed PD (n = 34), as well as control subjects (n = 30), underwent skin punch biopsy at multiple sites (neck, lower back, thigh, and lower leg). The skin biopsy samples (198 in total) were divided in half to be analyzed by RT-QuIC assay in two independent laboratories. The α-synuclein RT-QuIC assay of multiple skin biopsies supported the clinical diagnosis of PD with a diagnostic accuracy of 88.9% and showed a high degree of inter-rater agreement between the two laboratories (92.2%). Higher α-synuclein seeding activity in RT-QuIC was shown in patients with longer disease duration and more advanced disease stage and correlated with the presence of REM sleep behavior disorder, cognitive impairment, and constipation. The α-synuclein RT-QuIC assay of minimally invasive skin punch biopsy is a reliable and reproducible biomarker for Parkinson's disease. Moreover, α-synuclein RT-QuIC seeding activity in the skin may serve as a potential indicator of progression as it correlates with the disease stage and certain non-motor symptoms.
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BACKGROUND: Interactions between the gut microbiota, microglia, and aging may modulate Alzheimer's disease (AD) pathogenesis but the precise nature of such interactions is not known. METHODS: We developed an integrated multi-dimensional, knowledge-driven, systems approach to identify interactions among microbial metabolites, microglia, and AD. Publicly available datasets were repurposed to create a multi-dimensional knowledge-driven pipeline consisting of an integrated network of microbial metabolite-gene-pathway-phenotype (MGPPN) consisting of 34,509 nodes (216 microbial metabolites, 22,982 genes, 1329 pathways, 9982 mouse phenotypes) and 1,032,942 edges. RESULTS: We evaluated the network-based ranking algorithm by showing that abnormal microglia function and physiology are significantly associated with AD pathology at both genetic and phenotypic levels: AD risk genes were ranked at the top 6.4% among 22,982 genes, P < 0.001. AD phenotypes were ranked at the top 11.5% among 9982 phenotypes, P < 0.001. A total of 8094 microglia-microbial metabolite-gene-pathway-phenotype-AD interactions were identified for top-ranked AD-associated microbial metabolites. Short-chain fatty acids (SCFAs) were ranked at the top among prioritized AD-associated microbial metabolites. Through data-driven analyses, we provided evidence that SCFAs are involved in microglia-mediated gut-microbiota-brain interactions in AD at both genetic, functional, and phenotypic levels. CONCLUSION: Our analysis produces a novel framework to offer insights into the mechanistic links between gut microbial metabolites, microglia, and AD, with the overall goal to facilitate disease mechanism understanding, therapeutic target identification, and designing confirmatory experimental studies.
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Enfermedad de Alzheimer , Microbioma Gastrointestinal , Enfermedad de Alzheimer/genética , Animales , Encéfalo , Ratones , Microglía , FenotipoRESUMEN
Definitive diagnosis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.
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Ensayos Analíticos de Alto Rendimiento/métodos , Enfermedad por Cuerpos de Lewy/diagnóstico , Enfermedad de Parkinson/diagnóstico , alfa-Sinucleína/análisis , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
IMPORTANCE: Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown. OBJECTIVE: To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS: This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020. MAIN OUTCOMES AND MEASURES: Skin αSynP seeding activity was analyzed by RT-QuIC and PMCA assays. RESULTS: A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynP seeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA. CONCLUSIONS AND RELEVANCE: This study provides proof-of-concept that skin αSynP seeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.
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Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.
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Cobamidas/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Vitamina B 12/análogos & derivados , Complejo Vitamínico B/farmacología , Regulación Alostérica , Animales , Caenorhabditis elegans , Modelos Animales de Enfermedad , Drosophila melanogaster , Reposicionamiento de Medicamentos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , RatasRESUMEN
INTRODUCTION: LRRK2 G2019S mutation carriers with Parkinson's disease (PD) have been generally indistinguishable from those with idiopathic PD, with the exception of variable differences in some motor and non-motor domains, including cognition, gait, and balance. LRRK2 G2019S is amongst the most common genetic etiologies for PD, particularly in Ashkenazi Jewish (AJ) populations. METHODS: This cross-sectional data collection study sought to clarify the phenotype of LRRK2 G2019S mutation carriers with PD. Primary endpoints were the Movement Disorder Society Unified Parkinson Disease Rating Scale (MDS-UPDRS) and Montreal Cognitive Assessment (MoCA). Other motor and non-motor data were also assessed. The Mann-Whitney U Test was utilized to compare LRRK2 G2019S carriers with PD (LRRK2+) with non-carrier PD controls who were matched for age, gender, education, and PD duration. Survival analyses and log rank tests were utilized to compare interval from onset of PD to development of motor and non-motor complications. RESULTS: We screened 251 subjects and 231 completed the study, of whom 9 were LRRK2+, including 7 AJ subjects. 22.73% of AJ subjects with a family history of PD (FH) and 12.96% of AJ subjects without a FH were LRRK2+. There were no significant differences between the 9 LRRK2+ subjects and 19 matched PD controls in MDS-UPDRS, MoCA, or other motor and non-motor endpoints. CONCLUSION: Prevalence of the LRRK2 G2019S mutation in AJ and non-AJ subjects in our study population in Cleveland, Ohio was comparable to other clinical studies. There were no significant motor or non-motor differences between LRRK2+ PD and matched PD controls.
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Predisposición Genética a la Enfermedad , Heterocigoto , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación , Enfermedad de Parkinson/genética , Anciano , Estudios Transversales , Femenino , Humanos , Judíos/genética , Masculino , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/fisiopatología , Fenotipo , Proyectos Piloto , PrevalenciaRESUMEN
Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles (NFTs), senile plaques (SPs), and a progressive loss of neuronal cells in selective brain regions. Rab10, a small Rab GTPase involved in vesicular trafficking, has recently been identified as a novel protein associated with AD. Interestingly, Rab10 is a key substrate of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine protein kinase genetically associated with the second most common neurodegenerative disease Parkinson's disease. However, the phosphorylation state of Rab10 has not yet been investigated in AD. Here, using a specific antibody recognizing LRRK2-mediated Rab10 phosphorylation at the amino acid residue threonine 73 (pRab10-T73), we performed immunocytochemical analysis of pRab10-T73 in hippocampal tissues of patients with AD. pRab10-T73 was prominent in NFTs in neurons within the hippocampus in all cases of AD examined, whereas immunoreactivity was very faint in control cases. Other characteristic AD pathological structures including granulovacuolar degeneration, dystrophic neurites and neuropil threads also contained pRab10-T73. The pRab10-T73 immunoreactivity was diminished greatly following dephosphorylation with alkaline phosphatase. pRab10-T73 was further found to be highly co-localized with hyperphosphorylated tau (pTau) in AD, and demonstrated similar pathological patterns as pTau in Down syndrome and progressive supranuclear palsy. Although pRab10-T73 immunoreactivity could be noted in dystrophic neurites surrounding SPs, SPs were largely negative for pRab10-T73. These findings indicate that Rab10 phosphorylation could be responsible for aberrations in the vesicle trafficking observed in AD leading to neurodegeneration.
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Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al GTP rab/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/patología , Fosforilación , Placa Amiloide/patología , Treonina/metabolismoRESUMEN
In most human sporadic prion diseases the phenotype is consistently associated with specific pairings of the genotype at codon 129 of the prion protein gene and conformational properties of the scrapie PrP (PrPSc) grossly identified types 1 and 2. This association suggests that the 129 genotype favours the selection of a distinct strain that in turn determines the phenotype. However, this mechanism cannot play a role in the phenotype determination of sporadic fatal insomnia (sFI) and a subtype of sporadic Creutzfeldt-Jakob disease (sCJD) identified as sCJDMM2, which share 129 MM genotype and PrPSc type 2 but are associated with quite distinct phenotypes. Our detailed comparative study of the PrPSc conformers has revealed major differences between the two diseases, which preferentially involve the PrPSc component that is sensitive to digestion with proteases (senPrPSc) and to a lesser extent the resistant component (resPrPSc). We conclude that these variations are consistent with two distinct strains in sFI and sCJDMM2, and that the rarer sFI is the result of a variant strain selection pathway that might be favoured by a different brain site of initial PrPSc formation in the two diseases.
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Enfermedades por Prión/clasificación , Proteínas Priónicas/genética , Priones/clasificación , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Genotipo , Glicosilación , Humanos , Insomnio Familiar Fatal/genética , Insomnio Familiar Fatal/metabolismo , Fenotipo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Proteínas Priónicas/metabolismo , Priones/genéticaRESUMEN
Misfolded alpha-synuclein (AS) and other neurodegenerative disorder proteins display prion-like transmission of protein aggregation. Factors responsible for the initiation of AS aggregation are unknown. To evaluate the role of amyloid proteins made by the microbiota we exposed aged rats and transgenic C. elegans to E. coli producing the extracellular bacterial amyloid protein curli. Rats exposed to curli-producing bacteria displayed increased neuronal AS deposition in both gut and brain and enhanced microgliosis and astrogliosis compared to rats exposed to either mutant bacteria unable to synthesize curli, or to vehicle alone. Animals exposed to curli producing bacteria also had more expression of TLR2, IL-6 and TNF in the brain than the other two groups. There were no differences among the rat groups in survival, body weight, inflammation in the mouth, retina, kidneys or gut epithelia, and circulating cytokine levels. AS-expressing C. elegans fed on curli-producing bacteria also had enhanced AS aggregation. These results suggest that bacterial amyloid functions as a trigger to initiate AS aggregation through cross-seeding and also primes responses of the innate immune system.
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Amiloide/farmacología , Proteínas Bacterianas/farmacología , Caenorhabditis elegans/metabolismo , Proteínas de Escherichia coli/farmacología , Escherichia coli , Agregación Patológica de Proteínas/inducido químicamente , Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Ratas , Ratas Endogámicas F344RESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.
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Glutarredoxinas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Línea Celular Tumoral , Cisteína/metabolismo , Glutatión/metabolismo , Humanos , Ratones , Proteína Desglicasa DJ-1/química , Proteína Desglicasa DJ-1/deficiencia , Procesamiento Proteico-PostraduccionalRESUMEN
Parkinson's disease (PD) results from the loss of dopaminergic neurons in the substantia nigra portion of the midbrain, and represents the second most common neurodegenerative disease in the world. Although the etiology of PD is currently unclear, oxidative stress and redox dysfunction are generally understood to play key roles in PD pathogenesis and progression. Aging and environmental factors predispose cells to adverse effects of redox changes. In addition to these factors, genetic mutations linked to PD have been observed to disrupt the redox balance. Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with autosomal dominant PD, and several of these mutations have also been shown to increase the levels of reactive oxygen species in cells. Anti-oxidant proteins are necessary to restore the redox balance and maintain cell viability. Over the past decade studies have started to demonstrate the critical importance for redox proteins mediating neuronal protection in models of PD. This commentary briefly describes some of the factors hypothesized to contribute to PD, specifically regarding the redox changes that occur in PD. Dysregulation of redox proteins in PD is highlighted by some of the work detailing the roles of peroxiredoxin-3 and thioredoxin-1 in models of PD. In an attempt to generate novel therapies for PD, several potent inhibitors of LRRK2 have been developed. The use of these compounds, both as tools to understand the biology of LRRK2 and as potential therapeutic strategies is also discussed. This mini-review then provides a historical prospective on the discovery and characterization of glutaredoxin (Grx1), and briefly describes current understanding of the role of Grx1 in PD. The review concludes by highlighting our recent publication describing the novel role for Grx1 in mediating dopaminergic neuronal protection both in vitro and in vivo.
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Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , FMN Reductasa/metabolismo , Proteínas PrPC/fisiología , Animales , Transporte Biológico , Células Hep G2 , Humanos , Hierro/metabolismo , Hígado/metabolismo , Ratones NoqueadosRESUMEN
Parkinson's disease (PD) is characterized by selective degeneration of dopaminergic neurons. Although the etiology of PD remains incompletely understood, oxidative stress has been implicated as an important contributor in the development of PD. Oxidative stress can lead to oxidation and functional perturbation of proteins critical to neuronal survival. Glutaredoxin 1 (Grx1) is an evolutionally conserved antioxidant enzyme that repairs protein oxidation by reversing the oxidative modification of cysteine known as S-glutathionylation. We aimed to explore the regulatory role of Grx1 in PD. We first examined the levels of Grx1 in postmortem midbrain samples from PD patients, and observed that Grx1 content is decreased in PD, specifically within the dopaminergic neurons. We subsequently investigated the potential role of Grx1 deficiency in PD pathogenesis by examining the consequences of loss of the Caenorhabditis elegans Grx1 homolog in well-established worm models of familial PD caused by overexpression of pathogenic human LRRK2 mutants G2019S or R1441C. We found that loss of the Grx1 homolog led to significant exacerbation of the neurodegenerative phenotype in C. elegans overexpressing the human LRRK2 mutants. Re-expression in the dopaminergic neurons of the active, but not a catalytically inactive form of the Grx1 homolog rescued the exacerbated phenotype. Loss of the Grx1 homolog also exacerbated the neurodegenerative phenotype in other C. elegans models, including overexpression of human α-synuclein and overexpression of tyrosine hydroxylase (a model of sporadic PD). Therefore, our results reveal a novel neuroprotective role of glutaredoxin against dopaminergic neurodegeneration in models of familial and sporadic PD.
Asunto(s)
Caenorhabditis elegans/genética , Glutarredoxinas/genética , Proteínas del Helminto/metabolismo , Enfermedad de Parkinson/genética , Animales , Supervivencia Celular , Cisteína/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Glutarredoxinas/deficiencia , Glutarredoxinas/metabolismo , Proteínas del Helminto/genética , Homeostasis , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mesencéfalo/metabolismo , Estrés Oxidativo , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/toxicidad , Animales , Animales Modificados Genéticamente , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Mutación , Neuronas/citología , Neurotoxinas/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/orina , Priones/patogenicidad , Priones/orina , Animales , Bioensayo , Encéfalo , Humanos , Ratones , Ratones Transgénicos , Sensibilidad y EspecificidadRESUMEN
The leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of autosomal-dominant Parkinson disease (PD). Mitochondrial dysfunction represents a critical event in the pathogenesis of PD. We demonstrated that wild-type (WT) LRRK2 expression caused mitochondrial fragmentation along with increased mitochondrial dynamin-like protein (DLP1, also known as DRP1), a fission protein, which was further exacerbated by expression of PD-associated mutants (R1441C or G2019S) in both SH-SY5Y and differentiated primary cortical neurons. We also found that LRRK2 interacted with DLP1, and LRRK2-DLP1 interaction was enhanced by PD-associated mutations that probably results in increased mitochondrial DLP1 levels. Co-expression of dominant-negative DLP1 K38A or WT Mfn2 blocked LRRK2-induced mitochondrial fragmentation, mitochondrial dysfunction and neuronal toxicity. Importantly, mitochondrial fragmentation and dysfunction were not observed in cells expressing either GTP-binding deficient mutant LRRK2 K1347A or kinase-dead mutant D1994A which has minimal interaction with DLP1 and did not increase the mitochondrial DLP1 level. We concluded that LRRK2 regulates mitochondrial dynamics by increasing mitochondrial DLP1 through its direct interaction with DLP1, and LRRK2 kinase activity plays a critical role in this process.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Dinaminas , GTP Fosfohidrolasas/genética , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Neuronas/metabolismo , Neuronas/ultraestructura , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estrés Fisiológico , TransfecciónRESUMEN
Transmissible spongiform encephalopathies (TSEs) are a group of zoonotic and fatal neurodegenerative disorders that affect humans and animals. The pathogenesis of TSEs involves a conformational conversion of the cellular prion protein (PrP) into abnormal isoforms. Currently, cellular and pathological forms of PrP are differentiated using specific antibody-based analyses that are resource intensive and not applicable to all species and strains. Thus, there is an urgent need for sensitive and efficient assays that can detect pathological forms of PrP. Using systematic evolution of ligands by exponential enrichment, we developed DNA aptamers that can differentiate normal and abnormal PrP isoforms. These aptamers represent the first reagents that can identify pathological isoforms of PrP across multiple host species. Second, they are able to distinguish different strains of prions. Third, they can be used to detect prions in peripheral blood cells, which are otherwise undetectable using conventional antibody-based detection methods. Thus, DNA aptamers offer promise for the development of presymptomatic screens of tissue, blood and other body fluids for prion contamination.