HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.

The human brain acetylome reveals that decreased acetylation of mitochondrial proteins associates with Alzheimer's disease.

Metabolic changes that correlate to cognitive changes are well-known in Alzheimer's disease (AD). Metabolism is often linked to functional changes in proteins by post-translational modifications. The importance of the regulation of transcription by acetylation is well documented. Advanced mass spectrometry reveals hundreds of acetylated proteins in multiple tissues, but the acetylome of human brain, its functional significance, and the changes with disease are unknown. Filling this gap is critical for understanding the pathophysiology and development of therapies. To fill this gap, we assessed the human brain acetylome in human brain and its changes with AD. More than 5% of the 4,442 proteins from the human brain global proteome were acetylated. Acetylated proteins were primarily found in the cytosol (148), mitochondria (100), nucleus (91), and plasma membrane (58). The comparison of the brain acetylome in controls to that of patients with AD revealed striking and selective differences in terms of its abundances of acetylated peptides/sites. Acetylation of 18 mitochondrial proteins decreased, while acetylation of two cytosolic proteins, tau and GFAP, increased. Our experiments demonstrate that acetylation at some specific lysine sites alters enzyme function. The results indicate that general activation of de-acetylases (i.e., sirtuins) is not an appropriate therapeutic approach for AD.

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human-induced pluripotent stem cells, neural stem cells, and neurons.

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC-derived neurons. Inhibition of KGDHC for one or 24 hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD.