Cholesterol alters mitophagy by impairing optineurin recruitment and lysosomal clearance in Alzheimer's disease.

BACKGROUND: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer's disease (AD) pathogenesis. Amyloid-beta (Aß) 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 Aß 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 Aß 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.

Oxidative inactivation of amyloid beta-degrading proteases by cholesterol-enhanced mitochondrial stress.

Familial early-onset forms of Alzheimer's disease (AD) are linked to overproduction of amyloid beta (Aß) peptides, while decreased clearance of Aß is the driving force leading to its toxic accumulation in late-onset (sporadic) AD. Oxidative modifications and defective function have been reported in Aß-degrading proteases such as neprilysin (NEP) and insulin-degrading enzyme (IDE). However, the exact mechanisms that regulate the proteolytic clearance of Aß and its deficits are largely unknown. We have previously showed that cellular cholesterol loading, by depleting the mitochondrial GSH (mGSH) content, stimulates Αß-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-ß-cyclodextrin. The recovery of the proteolytic activity after treatment was accompanied with a significant reduction of Aß levels. Supporting these results, cholesterol-enriched SH-SY5Y cells were more sensitive to Aß-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 Aß on intracellular AßDPs activities but also had an impact on extracellular IDE levels and function, stimulating the extracellular Aß 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 Aß peptides and AD progression.

Cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion.

Macroautophagy/autophagy failure with the accumulation of autophagosomes is an early neuropathological feature of Alzheimer disease (AD) that directly affects amyloid beta (Aß) 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 mouse-human 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 Aß and endogenous MAPT (microtubule associated protein tau), and stimulated autophagy-dependent Aß secretion. Exacerbated Aß-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 Aß. 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-ß-cyclodextrin completely rescued these alterations, making it a potential therapeutic tool for AD.

Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma.

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.

Endoplasmic reticulum stress mediates amyloid ß neurotoxicity via mitochondrial cholesterol trafficking.

Disrupted cholesterol homeostasis has been reported in Alzheimer disease and is thought to contribute to disease progression by promoting amyloid ß (Aß) accumulation. In particular, mitochondrial cholesterol enrichment has been shown to sensitize to Aß-induced neurotoxicity. However, the molecular mechanisms responsible for the increased cholesterol levels and its trafficking to mitochondria in Alzheimer disease remain poorly understood. Here, we show that endoplasmic reticulum (ER) stress triggered by Aß promotes cholesterol synthesis and mitochondrial cholesterol influx, resulting in mitochondrial glutathione (mGSH) depletion in older age amyloid precursor protein/presenilin-1 (APP/PS1) mice. Mitochondrial cholesterol accumulation was associated with increased expression of mitochondrial-associated ER membrane proteins, which favor cholesterol translocation from ER to mitochondria along with specific cholesterol carriers, particularly the steroidogenic acute regulatory protein. In vivo treatment with the ER stress inhibitor 4-phenylbutyric acid prevented mitochondrial cholesterol loading and mGSH depletion, thereby protecting APP/PS1 mice against Aß-induced neurotoxicity. Similar protection was observed with GSH ethyl ester administration, which replenishes mGSH without affecting the unfolded protein response, thus positioning mGSH depletion downstream of ER stress. Overall, these results indicate that Aß-mediated ER stress and increased mitochondrial cholesterol trafficking contribute to the pathologic progression observed in old APP/PS1 mice, and that ER stress inhibitors may be explored as therapeutic agents for Alzheimer disease.

APP/PS1 mice overexpressing SREBP-2 exhibit combined Aß accumulation and tau pathology underlying Alzheimer's disease.

Current evidence indicates that excess brain cholesterol regulates amyloid-ß (Aß) deposition, which in turn can regulate cholesterol homeostasis. Moreover, Aß neurotoxicity is potentiated, in part, by mitochondrial glutathione (mGSH) depletion. To better understand the relationship between alterations in cholesterol homeostasis and Alzheimer's disease (AD), we generated a triple transgenic mice featuring sterol regulatory element-binding protein-2 (SREBP-2) overexpression in combination with APPswe/PS1ΔE9 mutations (APP/PS1) to examine key biochemical and functional characteristics of AD. Unlike APP/PS1 mice, APP/PS1/SREBP-2 mice exhibited early mitochondrial cholesterol loading and mGSH depletion. Moreover, ß-secretase activation and Aß accumulation, correlating with oxidative damage and neuroinflammation, were accelerated in APP/PS1/SREBP-2 mice compared with APP/PS1 mice. Triple transgenic mice displayed increased synaptotoxicity reflected by loss of synaptophysin and neuronal death, resulting in early object-recognition memory impairment associated with deficits in spatial memory. Interestingly, tau pathology was present in APP/PS1/SREBP-2 mice, manifested by increased tau hyperphosphorylation and cleavage, activation of tau kinases and neurofibrillary tangle (NFT) formation without expression of mutated tau. Importantly, in vivo treatment with the cell permeable GSH ethyl ester, which restored mGSH levels in APP/PS1/SREBP-2 mice, partially prevented the activation of tau kinases, reduced abnormal tau aggregation and Aß deposition, resulting in attenuated synaptic degeneration. Taken together, these results show that cholesterol-mediated mGSH depletion is a key event in AD progression, accelerating the onset of key neuropathological hallmarks of the disease. Thus, therapeutic approaches to recover mGSH may represent a relevant strategy in the treatment of AD.

Endothelin-dependent vasoconstriction in human uterine artery: application to preeclampsia.

BACKGROUND: Reduced uteroplacental perfusion, the initiating event in preeclampsia, is associated with enhanced endothelin-1 (ET-1) production which feeds the vasoconstriction of uterine artery. Whether the treatments of preeclampsia were effective on ET-1 induced contraction and could reverse placental ischemia is the question addressed in this study. We investigated the effect of antihypertensive drugs used in preeclampsia and of ET receptor antagonists on the contractile response to ET-1 on human uterine arteries. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed, ex vivo, on human uterine artery samples obtained after hysterectomy. We studied variations in isometric tension of arterial rings in response to the vasoconstrictor ET-1 and evaluated the effects of various vasodilators and ET-receptor antagonists on this response. Among antihypertensive drugs, only dihydropyridines were effective in blocking and reversing the ET-1 contractile response. Their efficiency, independent of the concentration of ET-1, was only partial. Hydralazine, alpha-methyldopa and labetalol had no effect on ET-1 induced contraction which is mediated by both ET(A) and ET(B) receptors in uterine artery. ET receptors antagonists, BQ-123 and BQ-788, slightly reduced the amplitude of the response to ET-1. Combination of both antagonists was more efficient, but it was not possible to reverse the maximal ET-1-induced contraction with antagonists used alone or in combination. CONCLUSION: Pharmacological drugs currently used in the context of preeclampsia, do not reverse ET-1 induced contraction. Only dihydropyridines, which partially relax uterine artery previously contracted with ET-1, might offer interesting perspectives to improve placental perfusion.