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Background: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer’s disease (AD) pathogenesis. Amyloid-beta (Aβ) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Aβ clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy. Methods: Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2. Results: In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Aβ while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages. Conclusions: Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD., This work was supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) under grants: RTI2018–095572-B-100 to A.C., RTI2018–095672-B-I00 to A.M., SAF2017–89791-R to R.T.; Fundació La Marató de TV3 (2014–0930 to A.C.); the Instituto de Salud Carlos III under grant PI19/01410 to M.M.; the Agencia de Gestió d’Ajuts Universitaris i de Recerca, Spain (2017_SGR_177); and the CERCA Programme from the Generalitat de Catalunya, Spain. C.dD. has a FPU fellowship from Ministerio de Ciencia, Innovación y Universidades, Spain. We acknowledge support of the publication fee by the CSIC Open Acess Publication Support Initiative through its Unit of Information Resource for Research (URICI).

Familial early-onset forms of Alzheimer's disease (AD) are linked to overproduction of amyloid beta (Aβ) peptides, while decreased clearance of Aβ is the driving force leading to its toxic accumulation in late-onset (sporadic) AD. Oxidative modifications and defective function have been reported in Aβ-degrading proteases such as neprilysin (NEP) and insulin-degrading enzyme (IDE). However, the exact mechanisms that regulate the proteolytic clearance of Aβ and its deficits are largely unknown. We have previously showed that cellular cholesterol loading, by depleting the mitochondrial GSH (mGSH) content, stimulates Αβ-induced mitochondrial oxidative stress and promotes AD-like pathology in APP-PSEN1-SREBF2 mice. Here, using the same AD mouse model we examined whether cholesterol-enhanced mitochondrial oxidative stress affects NEP and IDE function. We found that brain extracts from APP-PSEN1-SREBF2 mice displayed increased presence of oxidatively modified forms of NEP and IDE, associated with impaired enzymatic activities. Both alterations were substantially recovered after an in vivo treatment with the cholesterol-lowering agent 2-hydroxypropyl-β-cyclodextrin. The recovery of the proteolytic activity after treatment was accompanied with a significant reduction of Aβ levels. Supporting these results, cholesterol-enriched SH-SY5Y cells were more sensitive to Aβ-induced impairment of IDE and NEP function in vitro. The rise of cellular cholesterol also stimulated the extracellular release of IDE by an unconventional autophagy-coordinated mechanism. Recovery of depleted pool of mGSH in these cells not only prevented the detrimental effect of Aβ on intracellular AβDPs activities but also had an impact on extracellular IDE levels and function, stimulating the extracellular Aβ degrading activity. Therefore, changes in brain cholesterol levels by modifying the mGSH content would play a key role in IDE and NEP-mediated proteolytic elimination of Aβ peptides and AD progression., Graphical abstract Image 1, Highlights • Cholesterol regulates IDE and NEP by enhancing the detrimental effect of Aβ on their proteolytic activities. • Cholesterol-mediated mitochondrial GSH depletion is responsible for the oxidative impairment of IDE and NEP. • High cholesterol levels induce the release of inactive IDE through secretory autophagy. • A rise in cellular cholesterol affects the extracellular Aβ degradation, favoring oligomers formation. • Cholesterol lowering compounds and antioxidant therapy restore IDE and NEP activity.

Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated., This study was funded by grants from the Instituto de Salud Carlos III (FIS PI12/00110, PI09/00056 to A.M., PI13/00374 to M.M, PI13/01339 to A.V., and SAF2015-69944-R and PI11/0325 to J.F.C.), Ministerio de Economía y Competitividad (SAF2012/34831 to J.F.C., SAF2014-57674-R to C.G.R. and SAF2013-47246-R to A.C.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases, US NIAAA to J.F.C.); Fundació la Marató de TV3 to J.F.C. and A.C., Mutua Madrileña (AP103502012) to C.G.R., and by CIBEREHD from the Instituto de Salud Carlos III. We also want to thank the support of the AGAUR (2014SGR785) from the Generalitat de Catalunya.

Macroautophagy/autophagy failure with the accumulation of autophagosomes is an early neuropathological feature of Alzheimer disease (AD) that directly affects amyloid beta (Ab) metabolism. Although loss of presenilin 1 function has been reported to impair lysosomal function and prevent autophagy flux, the detailed mechanism leading to autophagy dysfunction in AD remains to be elucidated. The resemblance between pathological hallmarks of AD and Niemann-Pick Type C disease, including endosome-lysosome abnormalities and impaired autophagy, suggests cholesterol accumulation as a common link. Using a mouse model of AD (APP-PSEN1-SREBF2 mice), expressing chimeric mousehuman amyloid precursor protein with the familial Alzheimer Swedish mutation (APP695swe) and mutant presenilin 1 (PSEN1-dE9), together with a dominant-positive, truncated and active form of SREBF2/SREBP2 (sterol regulatory element binding factor 2), we demonstrated that high brain cholesterol enhanced autophagosome formation, but disrupted its fusion with endosomal-lysosomal vesicles. The combination of these alterations resulted in impaired degradation of Ab and endogenous MAPT (microtubule associated protein tau), and stimulated autophagy-dependent Ab secretion. Exacerbated Ab-induced oxidative stress in APP-PSEN1-SREBF2 mice, due to cholesterol-mediated depletion of mitochondrial glutathione/mGSH, is critical for autophagy induction. In agreement, in vivo mitochondrial GSH recovery with GSH ethyl ester, inhibited autophagosome synthesis by preventing the oxidative inhibition of ATG4B deconjugation activity exerted by Ab. Moreover, cholesterol-enrichment within the endosomes-lysosomes modified the levels and membrane distribution of RAB7A and SNAP receptors (SNAREs), which affected its fusogenic ability. Accordingly, in vivo treatment with 2-hydroxypropyl-b-cyclodextrin completely rescued these alterations, making it a potential therapeutic tool for AD., This work was supported by Ministerio de Economía y Competitividad under Grant: SAF2013-47246-R to A.C., SAF2015-66515-R to A.M., SAF2015-69944-R to J.F-C.); FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); Fundació La Marató de TV3 (2014-0930); Instituto de Salud Carlos III under Grant PI13/00374 and PI16/00930 to M.M., US NIAAA under Center grant P50- AA-11999 from Research Center for Liver and Pancreatic Diseases to J.F. C. We also want to thank the support of the AGAUR (2014-SGR785) and CERCA Programme from the Generalitat de Catalunya. C.dD. has a FPU fellowship from Ministerio de Economía y Competitividad. This work was developed (in part) at the Centre Esther Koplowitz.