Promising Molecular Architectures for Two-Photon Probes in the Diagnosis of α-Synuclein Aggregates
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ABSTRACT:
SUBMITTER: Porcu S
PROVIDER: S-EPMC11207056 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
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Project description:Parkinson's disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence is increasing that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy. Our previous work has shown that pericytes-vascular mural cells that regulate the blood-brain barrier-contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes cleave fibrillar α-syn aggregates (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110), with cleaved α-syn remaining present for up to 21 days. The number of α-syn aggregates/cell and average aggregate size depends on the type of strain, but differences disappear within 5 five hours of treatment. Our results highlight the role brain vasculature may play in reducing α-syn aggregate burden in PD.
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Project description:The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.
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Project description:Parkinson's disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPP+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+. This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.
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Project description:The accumulation of proteinaceous deposits comprised largely of the α-synuclein protein is one of the main hallmarks of Parkinson's disease (PD) and related synucleinopathies. Their progressive development coincides with site-specific phosphorylation, oxidative stress and eventually, compromised neuronal function. However, modeling protein aggregate formation in animal or in vitro models has proven notably difficult. Here, we took advantage of a preclinical organotypic brain slice culture model to study α-synuclein aggregate formation ex vivo. We monitored the progressive and gradual changes induced by α-synuclein such as cellular toxicity, autophagy activation, mitochondrial dysfunction, cellular death as well as α-synuclein modification including site-specific phosphorylation. Our results demonstrate that organotypic brain slice cultures can be cultured for long periods of time and when cultured in the presence of aggregated α-synuclein, the molecular features of PD are recapitulated. Taken together, this ex vivo model allows for detailed modeling of the molecular features of PD, thus enabling studies on the cumulative effects of α-synuclein in a complex environment. This provides a platform to screen potential disease-modifying therapeutic candidates aimed at impeding α-synuclein aggregation and/or cellular transmission. Moreover, this model provides a robust replacement for in vivo studies that do not include behavioral experiments, thus providing a way to reduce the number of animals used in an accelerated timescale.
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Project description:Parkinson's disease (PD) is one of the most common neurodegenerative diseases. It is characterized pathologically by the aggregation of α-synuclein (αS) in the form of Lewy bodies and Lewy neurites. A major challenge in PD therapy is poor efficiency of drug delivery to the brain due to the blood-brain barrier (BBB). For this reason, nanomaterials, with significant advantages in drug delivery, have gained attention. On the other hand, recent studies have shown that nanoparticles can promote αS aggregation in salt solution. Therefore, we tested if nanoparticles could have the same effect in cell models. We found that nanoparticle can induce cells to form αS inclusions as shown in immunocytochemistry, and detergent-resistant αS aggregates as shown in biochemical analysis; and nanoparticles of smaller size can induce more αS inclusions. Moreover, the induction of αS inclusions is in part dependent on endolysosomal impairment and the affinity of αS to nanoparticles. More importantly, we found that the abnormally high level of endogenous lysosomotropic biomolecules (e.g., sphingosine), due to impairing the integrity of endolysosomes could be a determinant factor for the susceptibility of cells to nanoparticle-induced αS aggregation; and deletion of GBA1 gene to increase the level of intracellular sphingosine can render cultured cells more susceptible to the formation of αS inclusions in response to nanoparticle treatment. Ultrastructural examination of nanoparticle-treated cells revealed that the induced inclusions contained αS-immunopositive membranous structures, which were also observed in inclusions seeded by αS fibrils. These results suggest caution in the use of nanoparticles in PD therapy. Moreover, this study further supports the role of endolysosomal impairment in PD pathogenesis and suggests a possible mechanism underlying the formation of membrane-associated αS pathology.
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