RESUMEN
The cellular prion protein, PrPC, has been postulated to function as a receptor for α-synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of α-synuclein aggregates in neurodegenerative disorders such as Parkinson's disease. Previously, we generated the "Salt (S)" and "No Salt (NS)" strains of α-synuclein aggregates that cause distinct pathological phenotypes in M83 transgenic mice overexpressing A53T-mutant human α-synuclein. To test the hypothesis that PrPC facilitates the propagation of α-synuclein aggregates, we produced M83 mice that either express or do not express PrPC. Following intracerebral inoculation with the S or NS strain, the absence of PrPC in M83 mice did not prevent disease development and had minimal influence on α-synuclein strain-specified attributes such as the extent of cerebral α-synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced α-synuclein deposits, and the structural fingerprints of protease-resistant α-synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of α-synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of α-synuclein strains in mice that express A53T-mutant human α-synuclein. Thus, other putative receptors or cell-to-cell propagation mechanisms may have a larger effect on the spread of α-synuclein aggregates during disease.
Asunto(s)
Modelos Animales de Enfermedad , Ratones Transgénicos , Sinucleinopatías , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Ratones , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Humanos , Proteínas PrPC/metabolismo , Proteínas PrPC/genética , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
Neurodegenerative diseases represent a huge healthcare challenge which is predicted to increase with an aging population. Synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), present complex challenges in understanding their onset and progression. They are characterized by the abnormal aggregation of α-synuclein in the brain leading to neurodegeneration. Accumulating evidence supports the existence of distinct subtypes based on the site of α-synuclein aggregation initiation, genetics, and, more recently, neuroinflammation. Mediated by both central nervous system-resident cells, peripheral immune cells, and gut dysbiosis, neuroinflammation appears as a key process in the onset and progression of neuronal loss. Sex-based differences add another layer of complexity to synucleinopathies, influencing disease prevalence - with a known higher incidence of PD in males compared to females - as well as phenotype and immune responses. Biological sex affects neuroinflammatory pathways and the immune response, suggesting the need for sex-specific therapeutic strategies and biomarker identification. Here, we review the heterogeneity of synucleinopathies, describing the etiology, the mechanisms by which the inflammatory processes contribute to the pathology, and the consideration of sex-based differences to highlight the need for personalized therapeutics.
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Inflamación , Sinucleinopatías , alfa-Sinucleína , Humanos , Sinucleinopatías/inmunología , Sinucleinopatías/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/inmunología , Inflamación/inmunología , Animales , Femenino , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/etiología , Masculino , Factores Sexuales , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/metabolismo , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/patologíaRESUMEN
BACKGROUND: Parkinson's disease (PD) is a neurodegenerative condition characterized by the loss of dopaminergic neurons and the accumulation of Lewy-body protein aggregates containing misfolded α-synuclein (α-syn) in a phosphorylated form. The lack of effective models for drug screens has hindered drug development studies for PD. However, the recent development of in vitro brain-like organoids provides a new opportunity for evaluating therapeutic agents to slow the progression of this chronic disease. METHODS: In this study, we used a 3D brain-like organoid model to investigate the potential of repurposing Tilorone, an anti-viral drug, for impeding the propagation of α-synucleinopathy. We assessed the effect of Tilorone on the uptake of fluorescently labeled α-syn preformed fibrils (sPFF) and sPFF-induced apoptosis using confocal microscopy. We also examined Tilorone's impact on the phosphorylation of endogenous α-syn induced by pathogenic sPFF by immunoblotting midbrain-like organoid extracts. Additionally, quantitative RT-PCR and proteomic profiling of sPFF-treated organoids were conducted to evaluate the global impact of Tilorone treatment on tissue homeostasis in the 3D organoid model. RESULTS: Tilorone inhibits the uptake of sPFF in both mouse primary neurons and human midbrain-like organoids. Tilorone also reduces the phosphorylation of endogenous α-syn induced by pathogenic α-syn fibrils and mitigates α-syn fibril-induced apoptosis in midbrain-like organoids. Proteomic profiling of fibril-treated organoids reveals substantial alterations in lipid homeostasis by α-syn fibrils, which are reversed by Tilorone treatment. Given its safety profile in clinics, Tilorone may be further developed as a therapeutic intervention to alleviate the propagation of synucleinopathy in PD patients.
Asunto(s)
Mesencéfalo , Organoides , Sinucleinopatías , alfa-Sinucleína , Mesencéfalo/patología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Organoides/patología , Humanos , alfa-Sinucleína/metabolismo , Sinucleinopatías/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Modelos Biológicos , Apoptosis/efectos de los fármacos , Animales , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Ratones , ProteómicaRESUMEN
Synucleinopathies, including Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders characterized by the aberrant accumulation of α-synuclein (α-syn). Although no treatment is effective for synucleinopathies, the suppression of α-syn aggregation may contribute to the development of numerous novel therapeutic targets. Recent research revealed that nicotinic acetylcholine (nACh) receptor activation has neuroprotective effects and promotes the degradation of amyloid protein by activating autophagy. In an in vitro human-derived cell line model, we demonstrated that galantamine, the nAChR allosteric potentiating ligand, significantly reduced the cell number of SH-SY5Y cells with intracellular Lewy body-like aggregates by enhancing the sensitivity of α7-nAChR. In addition, galantamine promoted autophagic flux, and prevented the formation of Lewy body-resembled aggregates. In an in vivo synucleinopathy mouse model, the propagation of α-syn aggregation in the cerebral cortex was inhibited by galantamine administration for 90 days. These results suggest that α7-nAChR is expected to be a novel therapeutic target, and galantamine is a potential agent for synucleinopathies.
Asunto(s)
Autofagia , Galantamina , alfa-Sinucleína , Receptor Nicotínico de Acetilcolina alfa 7 , Galantamina/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Autofagia/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Sinucleinopatías/tratamiento farmacológico , Sinucleinopatías/metabolismo , Fármacos Neuroprotectores/farmacología , Masculino , Ratones , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/tratamiento farmacológico , Ratones Endogámicos C57BLRESUMEN
Disease-modifying strategies for Parkinson disease (PD), the most common synucleinopathy, represent a critical unmet medical need. Accumulation of the neuronal protein alpha-synuclein (αS) and abnormal lipid metabolism have each been implicated in PD pathogenesis. Here, we elucidate how retinoid-X-receptor (RXR) nuclear receptor signaling impacts these two aspects of PD pathogenesis. We find that activated RXR differentially regulates fatty acid desaturases, significantly reducing the transcript levels of the largely brain-specific desaturase SCD5 in human cultured neural cells and PD patient-derived neurons. This was associated with reduced perilipin-2 protein levels in patient neurons, reversal of αS-induced increases in lipid droplet (LD) size, and a reduction of triglyceride levels in human cultured cells. With regard to αS proteostasis, our study reveals that RXR agonism stimulates lysosomal clearance of αS. Our data support the involvement of Polo-like kinase 2 activity and αS S129 phosphorylation in mediating this benefit. The lowering of cellular αS levels was associated with reduced cytotoxicity. Compared to RXR activation, the RXR antagonist HX531 had the opposite effects on LD size, SCD, αS turnover, and cytotoxicity, all supporting pathway specificity. Together, our findings show that RXR-activating ligands can modulate fatty acid metabolism and αS turnover to confer benefit in cellular models of PD, including patient neurons. We offer a new paradigm to investigate nuclear receptor ligands as a promising strategy for PD and related synucleinopathies.
Asunto(s)
Metabolismo de los Lípidos , Lisosomas , Neuronas , Receptores X Retinoide , Transducción de Señal , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Humanos , Lisosomas/metabolismo , Neuronas/metabolismo , Neuronas/patología , Receptores X Retinoide/metabolismo , Receptores X Retinoide/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Células Cultivadas , Perilipina-2/metabolismo , Perilipina-2/genética , FosforilaciónRESUMEN
In this study, we develop and validate a new Parkinson's disease (PD) mouse model that can be used to better understand how the disease progresses and to test the effects of new, potentially disease-modifying, PD therapies. Our central hypothesis is that mitochondrial dysfunction intercalates with misfolded α-synuclein (α-syn) accumulation in a vicious cycle, leading to the loss of nigral neurons. Our hypothesis builds on the concept that PD involves multiple molecular insults, including mitochondrial dysfunction and aberrant α-syn handling. We predicted that mitochondrial deficits, due to heterozygous loss of Engrailed-1 (En1+/-), combined with bilateral injections of pathogenic α-syn fibrils (PFFs), will act to generate a highly relevant PD model - the En1/SYN model. Here, En1+/- mice received bilateral intrastriatal stereotaxic injections of either PBS or α-syn fibrils and were analyzed using automated behavioral tests and deep learning-assisted histological analysis at 2, 4, and 6 months post-injection. We observed significant and progressive Lewy body-like inclusion pathology in the amygdala, motor cortex, and cingulate cortex, as well as the loss of tyrosine hydroxylase-positive (TH+) cells in the substantia nigra. The En1/SYN model also exhibited significant motor impairments at 6 months post-injection, which were however not exacerbated as we had expected. Still, this model has a comprehensive number of PD-like phenotypes and is therefore superior when compared to the α-syn PFF or En1+/- models alone.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Homeodominio , Enfermedad de Parkinson , alfa-Sinucleína , Animales , Ratones , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/genética , Inteligencia Artificial , Sinucleinopatías/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/genética , Ratones Endogámicos C57BL , Masculino , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/efectos de los fármacos , HeterocigotoRESUMEN
In this issue of Neuron, Endo et al.1 develop a PET tracer capable of detecting alpha-synuclein (É-syn). With validation in animal models and humans, this tracer brings us closer to being able to monitor the synuclein aggregation process and associated pathological changes in Parkinson's disease (PD) and other synucleinopathies.
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Enfermedad de Parkinson , Tomografía de Emisión de Positrones , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Tomografía de Emisión de Positrones/métodos , Humanos , Animales , Sinucleinopatías/metabolismo , Sinucleinopatías/diagnóstico por imagen , Sinucleinopatías/patologíaRESUMEN
Aggregation of RNA binding proteins and dysregulation of RNA metabolism drives pathogenesis of multiple neurodegenerative diseases. In this issue of Neuron, Belur et al.1 identified pathological NONO/SFPQ inclusions and aberrant A-to-I-edited RNAs accumulated in nucleus, leading to dysregulation of gene expression and neurodegeneration in synucleinopathy-associated diseases.
Asunto(s)
Edición de ARN , Sinucleinopatías , Humanos , Sinucleinopatías/metabolismo , Sinucleinopatías/genética , Sinucleinopatías/patología , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Animales , ARN/genética , ARN/metabolismoRESUMEN
The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.
Asunto(s)
Cuerpos de Inclusión , Células Madre Pluripotentes Inducidas , alfa-Sinucleína , Células Madre Pluripotentes Inducidas/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Sinucleinopatías/genética , Neuronas/metabolismo , Neuronas/patología , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
PURPOSE OF REVIEW: Sleep disturbances are amongst most frequent non-motor symptoms of Parkinson's Disease (PD), and they are similarly frequently reported in other alpha-syncleinopathies, such as Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). More recently, the orexin system has been implicated in control of arousal based on salient environmental set points, and its dysregulation in sleep issues in alpha-synucleinopathies suggested by the findings from the translational animal models. However, its role in the patients with alpha-synucleinopathies remains unclear. We thus set to systematically review, and to critically assess, contemporary evidence on the association of the orexinergic system and sleep disturbances in alpha-synucleinopathies. In this systematic review, studies investigating orexin and sleep in alpha-synucleinopathies (Rapid Eye Movement (REM) Behaviour Disorder (RBD), Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA)) were identified using electronic database searches of PubMed, Web of Science and PsychINFO using MeSH terms, keywords, and title words such as "Alpha-synucleinopathies" AND "Orexin" AND "Sleep Disturbances". RECENT FINDINGS: 17 studies were included in this systemic review, of which 2 studies on RBD, 10 on PD, 4 on DLB, and 1 on MSA patients. Taken together, RBD and PD studies suggest a potential adaptive increase in orexin levels in early stages of the neurodegenerative process, with reduced levels more often reported for later, more advanced stages of illness. To date, no differences in orexin levels were demonstrated between MSA patients and healthy controls. There is a dearth of studies on the role of orexin levels in alpha-synucleinopathies. Moreover, significant methodologic limitations in the current body of work, including use of non-standardised research protocols and lack of prospective, multi-centre studies, disallow for any finite conclusion in regards to underlying pathomechanisms. Nonetheless, a picture of a complex, multifaceted relationship between the dysregulation of the orexinergic pathway and sleep disturbances in alpha-synucleinopathies is emerging. Hence, future studies disentangling orexinergic pathomechanisms of alpha-syncleinopathies are urgently needed to obtain a more comprehensive account of the role of orexinergic pathway in alpha-synucleinopathies. Pharmacological manipulations of orexins may have multiple therapeutic applications in treatment strategies, disease diagnosis, and might be effective for treating both motor and non-motor symptoms.
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Orexinas , Trastornos del Sueño-Vigilia , Sinucleinopatías , Animales , Humanos , Enfermedad por Cuerpos de Lewy/sangre , Enfermedad por Cuerpos de Lewy/complicaciones , Enfermedad por Cuerpos de Lewy/metabolismo , Atrofia de Múltiples Sistemas/sangre , Atrofia de Múltiples Sistemas/complicaciones , Atrofia de Múltiples Sistemas/metabolismo , Orexinas/sangre , Orexinas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/complicaciones , Trastornos del Sueño-Vigilia/sangre , Trastornos del Sueño-Vigilia/etiología , Trastornos del Sueño-Vigilia/metabolismo , Sinucleinopatías/sangre , Sinucleinopatías/complicaciones , Sinucleinopatías/metabolismoRESUMEN
Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are characterized by neuronal loss accompanied by α-synuclein (α-syn) accumulation in the brain. While research conventionally focused on brain pathology, there is growing interest in peripheral alterations. Erythrocytes, which are rich in α-syn, have emerged as a compelling site for synucleinopathies-related alterations. Erythrocyte-derived extracellular vesicles (EVs), containing pathological α-syn species, can traverse the blood-brain barrier (BBB) under certain conditions and the gastrointestinal tract, where α-syn and gut microbiota interact extensively. This review explores the accumulating evidence of erythrocyte involvement in synucleinopathies, as well as their potential in disease pathogenesis and diagnosis. Given their unique properties, erythrocytes and erythrocyte-derived EVs may also serve as an ideal therapeutic platform for treating synucleinopathies and beyond.
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Eritrocitos , Sinucleinopatías , alfa-Sinucleína , Humanos , Eritrocitos/metabolismo , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Animales , alfa-Sinucleína/metabolismo , Vesículas Extracelulares/metabolismo , Barrera Hematoencefálica/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/patología , Encéfalo/metabolismoRESUMEN
Synucleinopathies, including dementia with Lewy bodies (DLB), Parkinson's disease (PD), and multiple system atrophy (MSA), are characterized by the presence of α-synuclein (α-syn) aggregates in the central nervous system. Recent evidence suggests that the heterogeneity of synucleinopathies may be partly explained by the fact that patients may have different α-syn fibrillar polymorphs with structural differences. In this study, we identify nuclease resistant 2'fluoro-pyrimidine RNA aptamers that can differentially bind to structurally distinct α-syn fibrillar polymorphs. Moreover, we introduce a method, AptaFOOT-Seq, designed to rapidly assess the affinity of a mixture of these aptamers for different α-SYN fibrillar polymorphs using next-generation sequencing. Our findings reveal that the binding behavior of aptamers can be very different when they are tested separately or in the presence of other aptamers. In this case, competition and cooperation can occur, providing a higher level of information, which can be exploited to obtain specific 'footprints' for different α-Syn fibrillar polymorphs. Notably, these footprints can distinguish polymorphs obtained from patients with PD, DLB or MSA. This result suggests that aptaFOOT-Seq could be used for the detection of misfolded or abnormal protein conformations to improve the diagnosis of synucleinopathies.
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Aptámeros de Nucleótidos , Enfermedad de Parkinson , Sinucleinopatías , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Humanos , Aptámeros de Nucleótidos/química , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Sinucleinopatías/metabolismo , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/genética , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Unión Proteica , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
α-Synuclein (αS) is an abundant presynaptic protein that regulates neurotransmission. It is also a key protein implicated in a broad class of neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD) and Lewy body dementia (LBD). Pathological αS deposits in these diseases, Lewy bodies (LBs)/neurites (LNs), contain about 90% of αS in its phospho-serine129 (pS129) form. Therefore, pS129 is widely used as a surrogate marker of pathology. However, recent findings demonstrate that pS129 is also physiologically triggered by neuronal activity to positively regulate synaptic transmission. In this opinion article, we contrast the literature on pathological and physiological pS129, with a special focus on the latter. We emphasize that pS129 is ambiguous and knowledge about the context is necessary to correctly interpret changes in pS129.
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alfa-Sinucleína , alfa-Sinucleína/metabolismo , Humanos , Fosforilación/fisiología , Animales , Serina/metabolismo , Enfermedad de Parkinson/metabolismo , Transmisión Sináptica/fisiología , Sinucleinopatías/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain and the formation of intracellular protein aggregates known as Lewy bodies, of which a major component is the protein α-synuclein. Several studies have suggested that mitochondria play a central role in the pathogenesis of PD, encompassing both familial and sporadic forms of the disease. Mitochondrial dysfunction is attributed to bioenergetic impairment, increased oxidative stress, damage to mitochondrial DNA, and alteration in mitochondrial morphology. These alterations may contribute to improper functioning of the central nervous system and ultimately lead to neurodegeneration. The perturbation of mitochondrial function makes it a potential target, worthy of exploration for neuroprotective therapies and to improve mitochondrial health in PD. Thus, in the current review, we provide an update on mitochondria-based therapeutic approaches toward α-synucleinopathies in PD.
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Mitocondrias , Enfermedad de Parkinson , Sinucleinopatías , Humanos , Enfermedad de Parkinson/terapia , Enfermedad de Parkinson/metabolismo , Sinucleinopatías/metabolismo , Sinucleinopatías/terapia , Mitocondrias/metabolismo , Animales , alfa-Sinucleína/metabolismoRESUMEN
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
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Encéfalo , Ratones Endogámicos C57BL , alfa-Sinucleína , Animales , Humanos , Masculino , Ratones , Fosfatasa Alcalina/metabolismo , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ratones Transgénicos , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Agregado de Proteínas/fisiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/patologíaRESUMEN
The aggregation of α-synuclein (α-syn), associated with Parkinson's disease (PD) extends from the peripheral autonomic nervous system to the cerebral cortex, indicating a neural circuit-based mechanism of spread. However, recent studies, have proposed alternative propagation routes beyond neural pathways, including transmission via bodily fluids, such as the blood. This notion expands our understanding of PD progression, underscoring the complexity of α-syn spread and its implications in disease management and therapeutic strategies.
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Enfermedad de Parkinson , Sinucleinopatías , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Sinucleinopatías/metabolismo , Enfermedad de Parkinson/metabolismo , AnimalesRESUMEN
Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of misfolded α-Synuclein (αSyn) in the brain. These conditions manifest with diverse clinical and pathophysiological characteristics. This disease diversity is hypothesized to be driven by αSyn strains with differing biophysical properties, potentially influencing prion-type propagation and consequentially the progression of illness. Previously, we investigated this hypothesis by injecting brain lysate (seeds) from deceased individuals with various synucleinopathies or human recombinant αSyn preformed fibrils (PFFs) into transgenic mice overexpressing either wild type or A53T human αSyn. In the studies herein, we expanded on these experiments, utilizing a panel of antibodies specific for the major carboxyl-terminally truncated forms of αSyn (αSynΔC). These modified forms of αSyn are found enriched in human disease brains to inform on potential strain-specific proteolytic patterns. With monoclonal antibodies specific for human αSyn cleaved at residues 103, 114, 122, 125, and 129, we demonstrate that multiple system atrophy (MSA) seeds and PFFs induce differing neuroanatomical spread of αSyn pathology associated with host specific profiles. Overall, αSyn cleaved at residue 103 was most widely present in the induced pathological inclusions. Furthermore, αSynΔC-positive inclusions were present in astrocytes, but more frequently in activated microglia, with patterns dependent on host and inoculum. These findings support the hypothesis that synucleinopathy heterogeneity might stem from αSyn strains with unique biochemical properties that include proteolytic processing, which could result in dominant strain properties.
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Encéfalo , Modelos Animales de Enfermedad , Ratones Transgénicos , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/inmunología , Animales , Humanos , Ratones , Encéfalo/patología , Encéfalo/metabolismo , Sinucleinopatías/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/inmunología , Anticuerpos Monoclonales , Atrofia de Múltiples Sistemas/patología , Atrofia de Múltiples Sistemas/inmunología , Atrofia de Múltiples Sistemas/metabolismo , Priones/inmunología , Priones/metabolismo , FemeninoRESUMEN
OBJECTIVE: Evidence of abnormal α-synuclein (α-Syn) deposition in the brain is required for definitive diagnosis of synucleinopathies, which remains challenging. The seed amplification assay (SAA) is an innovative technique that can detect the seeding activity of misfolded α-Syn, enabling the amplification and detection of minute quantities of pathogenic α-Syn aggregates. This study aimed to evaluate oral mucosa α-Syn SAA as possible diagnostic and prodromal biomarkers for synucleinopathies. METHODS: A total of 107 Parkinson's disease (PD) patients, 99 multiple system atrophy (MSA) patients, 33 patients with isolated rapid eye movement sleep behavior disorder (iRBD) and 103 healthy controls (HC) were included. The SAA was applied to detect the seeding activity of α-Syn from oral mucosa. A combination of morphological, biochemical, and biophysical methods was also used to analyze the fibrils generated from the oral mucosa α-Syn SAA. RESULTS: Structured illumination microscopy images revealed the increased α-Syn species in oral mucosa of PD, MSA, and iRBD patients than in HCs. Oral mucosa α-Syn SAA distinguished patients with PD from HC with 67.3% sensitivity and 90.3% specificity. Oral mucosa was α-Syn SAA positive in 53.5% MSA patients and 63.6% iRBD patients. Furthermore, the α-Syn fibrils generated from MSA demonstrated greater resistance to proteinase K digestion and exhibited stronger cytotoxicity compared to those from PD patients. CONCLUSION: Oral mucosa α-Syn seeding activity may serve as novel non-invasive diagnostic and prodromal biomarkers for synucleinopathies. The α-Syn aggregates amplified from the oral mucosa of PD and MSA exhibited distinct biochemical and biophysical properties. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Asunto(s)
Mucosa Bucal , Atrofia de Múltiples Sistemas , Enfermedad de Parkinson , Trastorno de la Conducta del Sueño REM , Sinucleinopatías , alfa-Sinucleína , Humanos , Trastorno de la Conducta del Sueño REM/metabolismo , Trastorno de la Conducta del Sueño REM/diagnóstico , alfa-Sinucleína/metabolismo , Femenino , Masculino , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/diagnóstico , Persona de Mediana Edad , Anciano , Sinucleinopatías/metabolismo , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/diagnóstico , Atrofia de Múltiples Sistemas/genética , Atrofia de Múltiples Sistemas/patología , Biomarcadores/metabolismoRESUMEN
αSynuclein (αSyn) misfolding and aggregation frequently precedes neuronal loss associated with Parkinson's Disease (PD) and other Synucleinopathies. The progressive buildup of pathological αSyn species results from alterations on αSyn gene and protein sequence, increased local concentrations, variations in αSyn interactome and protein network. Therefore, under physiological conditions, it is mandatory to regulate αSyn proteostasis as an equilibrium among synthesis, trafficking, degradation and extracellular release. In this frame, a crucial parameter is protein half-life. It provides indications of the turnover of a specific protein and depends on mRNA synthesis and translation regulation, subcellular localization, function and clearance by the designated degradative pathways. For αSyn, the molecular mechanisms regulating its proteostasis in neurons have been extensively investigated in various cellular models, either using biochemical or imaging approaches. Nevertheless, a converging estimate of αSyn half-life has not emerged yet. Here, we discuss the challenges in studying αSyn proteostasis under physiological and pathological conditions, the advantages and disadvantages of the experimental strategies proposed so far, and the relevance of determining αSyn half-life from a translational perspective.
Asunto(s)
alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Semivida , Animales , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Proteostasis/fisiología , Neuronas/metabolismoRESUMEN
Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.