Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

Abstract

Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases.

A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles

“…Recently the regulation of V-ATPase localization by PPT1 was described in neurons (27). The amino acid-dependent physical interaction between vATPase and the Ragulator complex facilitates the lysosomal recruitment of mTORC1 by Rag GTPases (28).…”

Cln3‐mutations underlying juvenile neuronal ceroid lipofuscinosis cause significantly reduced levels of Palmitoyl‐protein thioesterases‐1 (Ppt1)‐protein and Ppt1‐enzyme activity in the lysosome

“…We sought to determine the cause of increased lysosomal pH in JNCL fibroblasts. Previously, we reported that Ppt1‐deficiency leads to the misrouting of the V0a1 subunit of the v‐ATPase to the plasma membrane dysregulating lysosomal pH in cultured neurons from Cln1 −/− mice . Thus, we sought to determine whether Cln3 ‐mutations causing PPT1 insufficiency in cultured JNCL fibroblasts V0a1 is mislocalized to the plasma membrane and not to its normal location on lysosomal membrane.…”

“…Lysosomal acidification is regulated by v‐ATPase, a multi‐subunit protein localized to the lysosomal membrane. Previously, we reported that in Cln1 −/− mice, which mimic INCL and in cultured fibroblasts from INCL patients, PPT1‐deficiency causes misrouting of a critical subunit of v‐ATPase, V0a1, to the plasma membrane instead of its normal location to the lysosomal membrane dysregulating lysosomal acidification . Thus, we first sought to determine whether lysosomal pH is dysregulated in cultured JNCL patient fibroblasts.…”

“…Then the fibroblasts were labeled with 1 mM pH‐sensitive lysosensor green DND‐189 (L‐7535; Life Technologies‐Thermo Fisher Scientific) for 10 minutes at 37°C, washed with PBS (two to three times), then imaged using a fluorescein isothiocyanate channel in FluoroBrite DMEM media. To measure the lysosomal pH, we used Oregon green dextran (D‐7172; Life Technologies) and TMR dextran (D‐1819; Life Technologies (Thermo Fisher Scientific), as previously reported with minor modifications . Briefly, the lysosomal pH was measured by using the fluorescence ratio between the pH‐sensitive Oregon green (ex‐496, em‐524) and pH insensitive TMR (ex‐555, em‐580).…”

Fas‐apoptotic inhibitory molecule 2 localizes to the lysosome and facilitates autophagosome‐lysosome fusion through the LC3 interaction region motif–dependent interaction with LC3

“…[45][46][47] The pathophysiological roles of FAIM2 in the nervous system would be of interest. Other molecules may substitute FAIM2 function in cells not expressing FAIM2.…”