Abstract
Parkinson’s Disease (PD) is the second most common neurodegenerative disease, affecting nearly 8.5 million people worldwide. Its current etiopathogenesis is caused by a multitude of etiopathogenetic mechanisms like oxidative stress, mitochondrial dysfunction, -synuclein misfolding, lewy body formation, and neuroinflammation among others. Current treatments primarily focus on Symptomatic therapy via countering dopamine depletion.
Isoform specific inhibition of Histone Deacetylase-6 (HDAC-6) has been a long contested therapeutic strategy for Parkinson’s Disease (PD). Previous in-vitro studies report that HDAC-6 inhibition causes nuclear localization of HDAC-6, proteasomal system dysfunction, increased protein aggregation, and apoptosis. On the contrary , most in-vivo studies show that HDAC-6 inhibition can have a protective effect against toxin-induced PD models via countering neuroinflammation, oxidative stress and microglial activation. This reflects a gap in research translation from traditional monolayer cell culture models to animal studies. This study aims to develop a midbrain Neurosphere model for modeling Parkinson’s disease using neural progenitor stem cells (NPCs), and then evaluating the effect of an isoform-specific Histone Deacetylase-6 (HDAC-6) inhibitor on the NPCs.
Spheroid culture was initially generated using 3 methods: Hanging drop technique, Rotary cell culture system (RCCS), and Ultra-low attachment (ULA) microplates. These three methods were compared using various metrics to determine which could best fit the requirement for the project. The ULA microplate method was ultimately used to generate & differentiate the NPC spheroids.Differentiation was confirmed by immunostaining for differentiation markers like Forkhead Box Protein 2(FOXA2), Dopamine Transporter (DAT) & G-protein coupled, Inwardly Rectifying Potassium channel receptor-2 (GIRK2). -synuclein containing preformed fibrils (PFFs) were used as a positive control for disease induction. Next, a standard resazurin assay was performed for determining the cytotoxicity of the selected HDAC-6 inhibitor, Tubacin.
The effect of Tubacin was characterized on the expression levels of the differentiation markers on the spheroids, using Immunocytochemistry (ICC). Tubacin did not significantly affect the levels of FOXA2 and GIRK2, but it did show a protective effect on the immunofluorescence of DAT against PFF-induced dopaminergic neuron toxicity.
Tubacin treatment also lowered the expression of -synuclein, which was quantified using sandwich ELISA. The findings were visually confirmed via the aggresome assay, which studies ‘aggresomes’ which are visual signs of protein misfolding and aggregation, a key cellular biomarker for PD. Tubacin treatment significantly reduced aggresome levels which upregulated in the patient cell line, as well as attenuated the damage caused by prolonged PFF-treatment. Our findings indicate that isoform specific of HDAC-6 have therapeutic application in Parkinson’s disease.