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1.
Proc Natl Acad Sci U S A ; 119(15): e2109617119, 2022 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-35353605

RESUMEN

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129­α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129­α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129­α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129­α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129­α-syn (WT­α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129­α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129­α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129­α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129­α-syn as a measure of efficacy in clinical trials.


Asunto(s)
Amiloide , Enfermedad por Cuerpos de Lewy , Enfermedad de Parkinson , Agregación Patológica de Proteínas , alfa-Sinucleína , Amiloide/metabolismo , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosforilación , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Serina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
2.
PNAS Nexus ; 1(5): pgac259, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36712380

RESUMEN

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

3.
PLoS One ; 16(10): e0252635, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34613964

RESUMEN

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.


Asunto(s)
Fosforilación/fisiología , Agregado de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Serina/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Dopamina/metabolismo , Cuerpos de Lewy/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo
4.
Acta Neuropathol Commun ; 9(1): 31, 2021 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-33632316

RESUMEN

Pain is a common non-motor symptom of Parkinson's disease (PD), with current limited knowledge of its pathophysiology. Here, we show that peripheral inoculation of mouse alpha-synuclein (α-Syn) pre-formed fibrils, in a transgenic mouse model of PD, elicited retrograde trans-synaptic spreading of α-Syn pathology (pSer129) across sensory neurons and dorsal nerve roots, reaching central pain processing regions, including the spinal dorsal horn and the projections of the anterolateral system in the central nervous system (CNS). Pathological peripheral to CNS propagation of α-Syn aggregates along interconnected neuronal populations within sensory afferents, was concomitant with impaired nociceptive response, reflected by mechanical allodynia, reduced nerve conduction velocities (sensory and motor) and degeneration of small- and medium-sized myelinated fibers. Our findings show a link between the transneuronal propagation of α-Syn pathology with sensory neuron dysfunction and neuropathic impairment, suggesting promising avenues of investigation into the mechanisms underlying pain in PD.


Asunto(s)
Neuralgia/etiología , Neuralgia/fisiopatología , Degeneración Retrógrada/patología , Degeneración Retrógrada/fisiopatología , Células Receptoras Sensoriales/patología , Sinucleinopatías/patología , Sinucleinopatías/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Enfermedad de Parkinson/fisiopatología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructura , Transmisión Sináptica , alfa-Sinucleína/metabolismo
5.
Acta Neuropathol Commun ; 7(1): 213, 2019 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-31856920

RESUMEN

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.


Asunto(s)
Hipocampo/fisiopatología , Neuronas/fisiología , Agregación Patológica de Proteínas/fisiopatología , Sinucleinopatías/fisiopatología , alfa-Sinucleína/fisiología , Animales , Axones/patología , Axones/fisiología , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/patología , Técnicas de Cultivo de Órganos , Sinucleinopatías/patología , alfa-Sinucleína/genética
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