Effect of Clemizole on Alpha-Synuclein-Preformed Fibrils-Induced Parkinson's Disease Pathology: A Pharmacological Investigation.

Parkinson's disease (PD) is a neurodegenerative disorder associated with mitochondrial dysfunctions and oxidative stress. However, to date, therapeutics targeting these pathological events have not managed to translate from bench to bedside for clinical use. One of the major reasons for the lack of translational success has been the use of classical model systems that do not replicate the disease pathology and progression with the same degree of robustness. Therefore, we employed a more physiologically relevant model involving alpha-synuclein-preformed fibrils (PFF) exposure to SH-SY5Y cells and Sprague Dawley rats. We further explored the possible involvement of transient receptor potential canonical 5 (TRPC5) channels in PD-like pathology induced by these alpha-synuclein-preformed fibrils with emphasis on amelioration of oxidative stress and mitochondrial health. We observed that alpha-synuclein PFF exposure produced neurobehavioural deficits that were positively ameliorated after treatment with the TRPC5 inhibitor clemizole. Furthermore, Clemizole also reduced p-alpha-synuclein and diminished oxidative stress levels which resulted in overall improvements in mitochondrial biogenesis and functions. Finally, the results of the pharmacological modulation were further validated using siRNA-mediated knockdown of TRPC5 channels, which also decreased p-alpha-synuclein expression. Together, the results of this study could be superimposed in the future for exploring the beneficial effects of TRPC5 channel modulation for other neurodegenerative disorders and synucleopathies.

Immunotherapy: An emerging treatment option for neurodegenerative diseases.

Accumulation of misfolded proteins and protein aggregates leading to degeneration of neurons is a hallmark of several neurodegenerative diseases. Therapy mostly relies on symptomatic relief. Immunotherapy offers a promising approach for the development of disease-modifying routes. Such strategies have shown remarkable results in oncology, and this promise is increasingly being realized for neurodegenerative diseases in advanced preclinical and clinical studies. This review highlights cases of passive and active immunotherapies in Parkinson's and Alzheimer's diseases. The reasons for success and failure, wherever available, and strategies to cross the blood-brain barrier, are discussed. The need for conditional modulation of the immune response is also reflected on.

Meclofenoxate Inhibits Aggregation of Alpha-synuclein in vitro.

BACKGROUND: α-Synuclein, a natively disordered protein, is a key component of Lewy bodies, the ubiquitinated protein aggregates which are the pathological hallmark of Parkinson's disease (PD). Meclofenoxate (centrophenoxine) is a nootropic drug which has shown beneficial therapeutic effects in various neuronal diseases. Administration of meclofenoxate enhanced levels of dopamine and improved motor function in animal models of Parkinson's disease (PD). Evidence suggested that dopamine interacts with and modulates α-synuclein aggregation. OBJECTIVE: The aim of this work was to investigate whether the observed positive effect of addition of meclofenoxate, a nootropic agent, on dopamine level, could be correlated with its effect on aggregation of α-synuclein. METHODS: Purification of recombinant human α-synuclein was performed by anion exchange chromatography. The purified protein was incubated in the absence and presence of meclofenoxate and was analyzed for aggregation by Thioflavin T fluorescence spectroscopy. Conformational changes in α-synuclein were monitored by fluorescence spectroscopy and fluorescence quenching studies using a neutral quencher. Secondary structure analysis of α-synuclein was monitored by circular dichroism spectroscopy. RESULTS: Recombinant human α-synuclein was expressed and purified by anion-exchange chromatography. Incubation of α-synuclein with meclofenoxate led to lowering aggregation in a concentration-dependent manner. Reduction in formation of oligomers was seen which suggested the formation of an off-pathway species which did not give rise to an aggregation-competent entity. Fluorescence quenching studies revealed that the additive distorted the native conformation of α- synuclein, leading to the formation of lower amounts of aggregation-prone species. CONCLUSION: In the presence of higher concentrations of meclofenoxate, α-synuclein undergoes a change in its conformation. This change is not dependent on the concentration of the additive. This non-native conformer promotes the formation of a species which does not undergo further aggregation. Our study provides a mechanistic explanation of the earlier observation that meclofenoxate has a beneficial effect on progression of PD in animal models.

Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway.

Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.