Biology subjects

Oueslati, A.

Publications and source records attributed to Oueslati, A..

4 recordsLinked to original sources

bioRxiv2026

Phosphorylation and lipid droplets drive the irreversible phase transition of α-Synuclein in living cells

Aberrant phase transitions of -synuclein (-Syn) condensates, the pathological hallmark of Parkinson disease (PD) and related synucleinopathies, are difficult to dissect in living cells due to limited tools for spatiotemporal control of intracellular phase behavior. Here, we use an optogenetic-based light-inducible protein aggregation (LIPA) system to drive -Syn liquid-liquid phase separation (LLPS) in mammalian HEK-293T cells and human iPSC-derived neurons (iNeurons). Blue-light stimulation of LIPA--Syn induces dynamic, liquid like biomolecular condensates that initially remain fully reversible, but gradually undergo condensate maturation into an irreversible, solid like state upon prolonged stimulation, consistent with a liquid to solid phase transition. This transition is accompanied by a robust accumulation of phosphatase-resistant Ser129-phosphorylated -synuclein (pS129), a hallmark biochemical feature of Lewy pathology in PD and related synucleinopathies. Mechanistically, PLK2-mediated phosphorylation at Ser129 promotes condensate stabilization and irreversibility, identifying the PLK2-pS129 axis as a key regulator of the conversion of dynamic -Syn condensates into persistent assemblies. Importantly, transient liquid-like -Syn condensates are efficiently cleared, primarily through the proteasomal degradation pathway. In contrast, mature solid-like condensates become refractory to degradation, resulting in proteostasis impairment and progressive intracellular -Syn accumulation. A major finding of this study is the identification of a strong link between -Syn condensate maturation and lipid droplet (LD) biology. The formation of irreversible -Syn condensates is associated with a marked increase in both LD abundance and size. Moreover, mature LIPA--Syn condensates exhibit extensive interactions with LDs, disrupting lipid homeostasis and increasing cellular vulnerability. These findings suggest that LDs are not merely passive bystanders but active participants in the pathological maturation of -Syn condensates. Collectively, our results provide mechanistic insight into the molecular and cellular processes that govern the transition of -Syn condensates from reversible liquid assemblies to pathogenic solid-like aggregates. They identify Ser129 phosphorylation and lipid droplet interactions as critical determinants of condensate fate and establish aberrant condensate maturation as a potential source of neurotoxic -Syn species that contribute to the pathogenesis of PD and other synucleinopathies.

neuroscience↗

bioRxiv2026

WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease

The aggregation of -synuclein (-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinsons disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding -SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of -SYN assembly in neurons. We found that the initiation and accumulation of -SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the -SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo -SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances -SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated -SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of -SYN oligomerization in living neurons and identify the WDR44--SYN interaction as a promising therapeutic target for reducing -SYN pathology and enabling early intervention in PD.

neuroscience↗

bioRxiv2025

Recapitulating Parkinson's pathology in human iPSC dopaminergic neurons reveals new mechanistic insights into Lewy body formation and heterogeneity.

The accumulation of alpha-synuclein (aSyn) into intraneuronal inclusions of heterogeneous morphology, known as Lewy bodies (LB), is one of the defining diagnostic features of Parkinsons disease (PD); yet, our understanding of the mechanisms underpinning their formation and heterogeneity remains incomplete. Here, we present a human isogenic iPSC-derived dopaminergic neuron (iDA) model that faithfully recapitulates the diverse biochemical, morphological, and ultrastructural features of LB neuropathology in PD. Unlike other iDA seeding models, our model does not rely on aSyn overexpression, mutations, or genetic engineering, making it a more physiologically relevant system for studying PD. We demonstrate that the iDA model accurately reproduces the temporal relationships between neuritic and cell-body aSyn pathology, recapitulating the full biochemical spectrum, post-translational modifications (PTM), and morphological diversity of aSyn aggregates found in human PD tissue. Moreover, our work provides critical insight into how different pathways to aSyn fibrillization and the complex interaction between aSyn fibrils and membranous organelles influence the morphological diversity of LB-like inclusions. This model represents a versatile platform for investigating the mechanisms of pathology formation, maturation, and neuronal dysfunction, as well as supporting the development of diagnostics that capture the diversity of aSyn pathology in PD and related synucleinopathies.

neuroscience↗

bioRxiv2024

The combination of optogenetic-induced protein aggregation and proximity biotinylation assays strongly implicates endolysosomal proteins in the early stages of α-synuclein aggregation

Alpha-synuclein (-syn) aggregation is a defining feature of Parkinsons disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding -syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the Light-Inducible Protein Aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify -syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 -syn-interacting proteins, including both established and novel candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of -syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.

neuroscience↗