Cilostazol restores autophagy flux in bafilomycin A1-treated, cultured cortical astrocytes through lysosomal reacidification: roles of PKA, zinc and metallothionein 3

Abstract

Cilostazol, a phosphodiesterase 3 inhibitor, reduces the amyloid-beta (Aβ) burden in mouse models of Alzheimer disease by as yet unidentified mechanisms. In the present study, we examined the possibility that cilostazol ameliorates lysosomal dysfunction. Astrocytes treated with bafilomycin A1 (BafA1) exhibited markedly reduced DND-189 and acridine orange (AO) fluorescence, indicating reduced lysosomal acidity. In both cases, BafA1-induced alkalization was reversed by addition of cilostazol, dibutyryl cAMP or forskolin. All three agents significantly increased free zinc levels in lysosomes, and addition of the zinc chelator TPEN abrogated lysosomal reacidification. These treatments did not raise free zinc levels or reverse BafA1-mediated lysosomal alkalization in metallothionein 3 (Mt3)-null astrocytes, indicating that the increases in zinc in astrocytes were derived mainly from Mt3. Lastly, in FITC-Aβ-treated astrocytes, cilostazol reversed lysosomal alkalization, increased cathepsin D activity, and reduced Aβ accumulation in astrocytes. Cilostazol also reduced mHtt aggregate formation in GFP-mHttQ74-expressing astrocytes. Collectively, our results present the novel finding that cAMP/PKA can overcome the v-ATPase blocking effect of BafA1 in a zinc-and Mt3-dependent manner. Accumulation of abnormal protein aggregates is a common pathological finding in a variety of neurodegenerative disorders, including Alzheimer disease (AD) and Parkinson disease (PD) 1,2. While initial studies focused on the mechanism by which protein aggregates are generated in a particular neurodegenerative disease, more recent studies have begun to ask questions relating to how formed protein aggregates are cleared in the central nervous system (CNS). This new direction may open up a broader path for finding potential treatments applicable to a number of protein aggregation-associated neurodegenerative diseases. One of the most discussed mechanisms in this context is macroautophagy, or simply autophagy 3-6. Whereas many misfolded proteins are degraded by the ubiquitin-proteasome system (UBS), large protein aggregates cannot be degraded by the UBS, and instead are cleared by autophagy. In this process, double-membrane-delimited autophagophores wrap around protein aggregates, resulting in the formation of autophagosomes, which then fuse with lysosomes. Digestion of the inner membrane of the autophagosome results in autolysosome formation, and lysosomal acidic hydrolases subsequently degrade protein aggregates. Hence, boosting autophagy may help catabolize protein aggregates that play pathogenic roles in neurodegenerative diseases. For instance, the autophagy-related protein beclin-1 is reported to be decreased in AD, which might lead to diminished autophagy 5,7-9. However, an increasing body of evidence indicates that instead of generalized defects in autophagy, lysosomal dysfunction that results in a decrease in autophagosome-lysosome fusion or autophagy arrest may be a more specific cause of the reduced autophagy flux 10-13. More specifically...

Roles for H+/K+‐ATPase and zinc transporter 3 in cAMP‐mediated lysosomal acidification in bafilomycin A1‐treated astrocytes

“…In our previous studies, we have demonstrated that increasing cAMP concentrations can markedly restore lysosomal acidity even in astrocytes treated with bafilomycin A1 (BafA1), a potent and selective v‐ATPase inhibitor (Kim, Seo, Kim, & Koh, 2020; Koh, Kim, Hwang, Kim, & Park, 2019). Considering that lysosomal free zinc levels increase concurrently with cAMP treatment in a TPEN‐dependent manner, it may be possible that proton pumps other than v‐ATPase, acting in concert with certain zinc transporters, may contribute to the regulation of lysosomal pH in such cases.…”

Human neural stem cells restore spatial memory in a transgenic Alzheimer’s disease mouse model by an immunomodulating mechanism

“…NCAM, sVCAM-1, and cathepsin D were uniquely elevated in hNSC-treated animals when compared to vehicle controls. This could be related to an enriched population of stem cells, as NCAM and sVCAM-1 are known to be related to neural stem cell survival, proliferation, and migration ( Shan, 2013 ; Klein et al, 2016 ), whereas increases in the protease cathepsin D are associated with improved amyloid beta clearance ( Gao et al, 2020 ; Kim et al, 2020 ). This effect could also be related to hNSC secretion of IGF-1, shown to promote survival of NCAM-expressing neural progenitors ( Gago et al, 2003 ; Monzo et al, 2013 ), which can mediate and induce cathepsin D expression ( Cappelletti et al, 1996 ; Wang et al, 2000 ).…”

Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly

“…Clioquinol, a zinc ionophore, induces autophagy in a zinc-dependent manner and contributes to the clearance of aggregated proteins in astrocytes and neurons ( Park et al, 2011 ). The addition of cilostazol, a phosphodiesterase (PDE) inhibitor, was found to restore autophagic flux blocked by bafilomycin A1 via lysosomal re-acidification in a zinc-dependent manner ( Kim et al, 2020 ). Koh et al also showed that the H + /K + -ATPase/ZnT3 complex is recruited to lysosomes in a cAMP-dependent manner and functions as an alternative proton pump for lysosomes when the V-ATPase function is downregulated by bafilomycin A1 ( Lee and Koh, 2021 ).…”