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

Ha Na Kim,Bo-Ra Seo,Hyunjin Kim,Jae-Young Koh

doi:10.1038/s41598-020-66292-3

Ha Na Kim, Bo-Ra Seo + Show 2 more

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https://doi.org/10.1038/s41598-020-66292-3

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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.

Highlights

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]
Consistent with its potent effect as a PDE inhibitor, cilostazol (10 μM) treatment for 1 hour markedly increased the level of cAMP in astrocytes
Addition of 10 μM cilostazol or 300 μM cAMP largely abrogated the bafilomycin A1 (BafA1)-induced changes in lysosomal pH (Fig. 1b)

Summary

Introduction

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). 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 fuse with lysosomes. Presenilin mutations result in hypofunction of v-ATPase, a lysosomal proton pump[14,15,16] Protein aggregates such as amyloid-beta (Aβ) and www.nature.com/scientificreports α-synuclein can shift the lysosome pH in a more alkaline direction. We examined whether cilostazol can reacidify lysosomes, even in the presence of the v-ATPase inhibitor BafA1, and whether changes in cytosolic/lysosomal free zinc levels are somehow involved in this process

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