Mechanistic Study on HSP90B1 Regulation of ClC-7 Ubiquitination to Restore Lysosomal Function in the Protection...

Fluorosis can induce cognitive dysfunction. The cognitive impairment caused by fluorosis is closely related to endoplasmic reticulum stress (ERS) and abnormal lysosomal acidification. However, the specific molecular mechanism is not known, and effective intervention strategies are lacking. Diterpene ginkgolides meglumine injection (DGMI) is used clinically as a neuroprotective agent, but whether it can inhibit ERS, regulate lysosomal acidification, and ameliorate fluorosis-induced cognitive dysfunction merits investigation.

We used the Morris water maze to detect the cognitive function of mice, evaluated the neuronal survival by Nissl staining, assessed the apoptotic status in hippocampal neurons of mice by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and observed the degree of synaptic plasticity damage using Golgi staining and transmission electron microscopy. In cell experiments, we used the OG488 acidophilic fluorescent probe to detect lysosomal acidity; detected the co-localization of HSP90B1 with the ER and the co-localization of the chloride channel, voltage-sensitive 7 (ClC-7) with lysosomes using immunofluorescence technology; employed co-immunoprecipitation to detect the binding of HSP90B1, ClC-7, and membrane-associated RING-CH protein 1 (MARCHF1) as well as the ubiquitination degradation level of ClC-7; used western blotting to measure expression of ERS-related proteins (PERK, IRE1α, ATF6), glycogen synthase kinase 3β, and mammalian target of rapamycin signals.

DGMI significantly improved cognitive dysfunction in C57BL/6 mice with fluorosis, inhibited hippocampal neuronal apoptosis, and improved synaptic plasticity damage. In cell experiments, the detection results of apoptosis and percent survival were consistent with those of animal experiments in that DGMI could alleviate sodium fluoride-induced ERS and lysosomal-acidification disorders in HT22 cells. DGMI downregulates the expression of the ER chaperone HSP90B1, weakens its interaction with ClC-7, thereby inhibiting the MARCHF1 E3 ligase-mediated ubiquitination and degradation of ClC-7, ultimately alleviating lysosomal acidification disorders and improving cognitive function.

DGMI may exert cognitive protective effects against fluorosis-induced cognitive impairment by regulating lysosomal acidification through the HSP90B1-ClC-7 axis, which also alleviates ERS, apoptosis, and other related neuropathologic injuries.