Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson\'s disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Classical swine fever virus (CSFV) is an enveloped positive-sense RNA virus belonging to the Flaviviridae family. The virus utilizes cellular lipids and manipulates host lipid metabolism to ensure its replication, especially during virus invasion and replication steps. Therefore, identification of the molecular lipid metabolism pathways that are suitable targets is critical for the development of anti-CSFV therapeutics. In this study, we screened the anti-CSFV activity of 12 compounds targeting synthesis of cholesterol and fatty acids, cholesterol esters, and cholesterol transport. We found that 25-hydroxycholesterol (25HC), a regulator of cholesterol metabolism and transport, has potent anti-CSFV activity. Mechanistically, we showed that 25HC inhibited CSFV proliferation by blocking the entry of virions into porcine alveolar macrophages (3D4/21) by decreasing cholesterol abundance in the plasma membrane through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Finally, we revealed that cholesterol 25-hydroxylase (CH25H), a redox enzyme that mediates 25HC production, also restricted CSFV infection via both enzyme activity-dependent and -independent mechanisms. Collectively, our results shed light on the mechanisms by which 25HC inhibits CSFV entry into cells and suggests a potential new therapeutic method against CSFV infection.

Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.