Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn.

BACKGROUND: ß-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear. METHODS: We generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7. RESULTS: Deletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density. CONCLUSIONS: These results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

GLA Mutations Suppress Autophagy and Stimulate Lysosome Generation in Fabry Disease.

Fabry disease (FD) is an X-linked recessive inheritance lysosomal storage disorder caused by pathogenic mutations in the GLA gene leading to a deficiency of the enzyme alpha-galactosidase A (α-Gal A). Multiple organ systems are implicated in FD, most notably the kidney, heart, and central nervous system. In our previous study, we identified four GLA mutations from four independent Fabry disease families with kidney disease or neuropathic pain: c.119C>A (p.P40H), c.280T>C (C94R), c.680G>C (p.R227P) and c.801+1G>A (p.L268fsX3). To reveal the molecular mechanism underlying the predisposition to Fabry disease caused by GLA mutations, we analyzed the effects of these four GLA mutations on the protein structure of α-galactosidase A using bioinformatics methods. The results showed that these mutations have a significant impact on the internal dynamics and structures of GLA, and all these altered amino acids are close to the enzyme activity center and lead to significantly reduced enzyme activity. Furthermore, these mutations led to the accumulation of autophagosomes and impairment of autophagy in the cells, which may in turn negatively regulate autophagy by slightly increasing the phosphorylation of mTOR. Moreover, the overexpression of these GLA mutants promoted the expression of lysosome-associated membrane protein 2 (LAMP2), resulting in an increased number of lysosomes. Our study reveals the pathogenesis of these four GLA mutations in FD and provides a scientific foundation for accurate diagnosis and precise medical intervention for FD.

Sulforaphane Inhibits Oxidative Stress and May Exert Anti-Pyroptotic Effects by Modulating NRF2/NLRP3 Signaling Pathway in Mycobacterium tuberculosis-Infected Macrophages.

Sulforaphane (SFN) is a natural isothiocyanate derived from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbage. SFN plays a crucial role in maintaining redox homeostasis by interacting with the active cysteine residues of Keap1, leading to the dissociation and activation of NRF2 in various diseases. In this study, our objective was to investigate the impact of SFN on oxidative stress and pyroptosis in Mycobacterium tuberculosis (Mtb)-infected macrophages. Our findings demonstrated that Mtb infection significantly increased the production of iNOS and ROS, indicating the induction of oxidative stress in macrophages. However, treatment with SFN effectively suppressed the expression of iNOS and COX-2 and reduced MDA and ROS levels, while enhancing GSH content as well as upregulating NRF2, HO-1, and NQO-1 expression in Mtb-infected RAW264.7 macrophages and primary peritoneal macrophages from WT mice. These results suggest that SFN mitigates oxidative stress by activating the NRF2 signaling pathway in Mtb-infected macrophages. Furthermore, excessive ROS production activates the NLRP3 signaling pathway, thereby promoting pyroptosis onset. Further investigations revealed that SFN effectively suppressed the expression of NLRP3, Caspase-1, and GSDMD, IL-1ß, and IL-18 levels, as well as the production of LDH, suggesting that it may exhibit anti-pyroptotic effects through activation of the NRF2 signaling pathway and reductions in ROS production during Mtb infection. Moreover, we observed that SFN also inhibited the expression of NLRP3, ASC, Caspase1, and IL-1ß along with LDH production in Mtb-infected primary peritoneal macrophages from NFR2-/- mice. This indicates that SFN can directly suppress NLRP3 activation and possibly inhibit pyroptosis initiation in an NRF2-independent manner. In summary, our findings demonstrate that SFN exerts its inhibitory effects on oxidative stress by activating the NRF2 signaling pathway in Mtb-infected macrophages, while it may simultaneously exert anti-pyroptotic properties through both NRF2-dependent and independent mechanisms targeting the NLRP3 signaling pathway.