Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson's disease.

Studying Parkinson's disease (PD) is complex due to a lack of cellular models mimicking key aspects of protein pathology. Here, we present a protocol for inducing and monitoring α-synuclein aggregation in living cells using optogenetics. We describe steps for plasmid transduction, biochemical validation, immunocytochemistry, and live-cell confocal imaging. These induced aggregates fulfill the cardinal features of authentic protein inclusions observed in PD-diseased brains and offer a tool to study the role of protein aggregation in neurodegeneration. For complete details on the use and execution of this protocol, please refer to Bérard et al.1.

Optimized protocol for the generation of functional human induced-pluripotent-stem-cell-derived dopaminergic neurons.

Generation of functional human dopaminergic (DA) neurons from human induced pluripotent stem cells (hiPSCs) is a crucial tool for modeling dopamine-related human diseases and cell replacement therapies. Here, we present a protocol to combine neuralizing transcription factor (NGN2) programming and DA patterning to differentiate hiPSCs into mature and functional induced DA (iDA) neurons. We describe steps from transduction of hiPSCs and neural induction through to differentiation and maturation of near-pure, fully functional iDA neurons within 3 weeks. For complete details on the use and execution of this protocol, please refer to Sheta et al. (2022).1.

Combining NGN2 programming and dopaminergic patterning for a rapid and efficient generation of hiPSC-derived midbrain neurons.

The use of human derived induced pluripotent stem cells (hiPSCs) differentiated to dopaminergic (DA) neurons offers a valuable experimental model to decorticate the cellular and molecular mechanisms of Parkinson's disease (PD) pathogenesis. However, the existing approaches present with several limitations, notably the lengthy time course of the protocols and the high variability in the yield of DA neurons. Here we report on the development of an improved approach that combines neurogenin-2 programming with the use of commercially available midbrain differentiation kits for a rapid, efficient, and reproducible directed differentiation of hiPSCs to mature and functional induced DA (iDA) neurons, with minimum contamination by other brain cell types. Gene expression analysis, associated with functional characterization examining neurotransmitter release and electrical recordings, support the functional identity of the iDA neurons to A9 midbrain neurons. iDA neurons showed selective vulnerability when exposed to 6-hydroxydopamine, thus providing a viable in vitro approach for modeling PD and for the screening of small molecules with neuroprotective proprieties.

Switch of serotonergic descending inhibition into facilitation by a spinal chloride imbalance in neuropathic pain.

Descending control from the brain to the spinal cord shapes our pain experience, ranging from powerful analgesia to extreme sensitivity. Increasing evidence from both preclinical and clinical studies points to an imbalance toward descending facilitation as a substrate of pathological pain, but the underlying mechanisms remain unknown. We used an optogenetic approach to manipulate serotonin (5-HT) neurons of the nucleus raphe magnus that project to the dorsal horn of the spinal cord. We found that 5-HT neurons exert an analgesic action in naïve mice that becomes proalgesic in an experimental model of neuropathic pain. We show that spinal KCC2 hypofunction turns this descending inhibitory control into paradoxical facilitation; KCC2 enhancers restored 5-HT-mediated descending inhibition and analgesia. Last, combining selective serotonin reuptake inhibitors (SSRIs) with a KCC2 enhancer yields effective analgesia against nerve injury-induced pain hypersensitivity. This uncovers a previously unidentified therapeutic path for SSRIs against neuropathic pain.

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

Alpha-Synuclein and the Endolysosomal System in Parkinson's Disease: Guilty by Association.

Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson's disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders.