HMGCS2 promotes autophagic degradation of the amyloid-ß precursor protein through ketone body-mediated mechanisms.

HMGCS2 (mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2) is a control enzyme in ketogenesis. The mitochondrial localization and interaction with APP (ß-amyloid precursor protein) suggest that HMGCS2 may play a role in the pathophysiology of AD (Alzheimer's disease). Here we report that overexpression of HMGCS2 decreased levels of APP and related CTFs (carboxy-terminal fragments), which was largely prevented by an autophagic inhibitor chloroquine. In addition, HMGCS2 enhancement of autophagic marker LC3II was diminished by rapamycin, an inhibitor of mechanistic target of rapamycin. Moreover, deprivation of EBSS (Earle's Balanced Salt Solution) significantly augmented the effect of HMGCS2 on LC3II, while acetoacetate reversed the reduction of LC3II, APP and CTFs which was induced by HMGCS2 knockdown. In the presence of acetoacetate, rapamycin failed to induce further increase of LC3II, which mimicked the effect of HMGCS2 overexpression. Finally, HMGCS2 enhanced the antioxidant response. Collectively, HMGCS2 shares with ketone bodies common features in autophagic clearance of APP and CTFs, suggesting that ketone bodies play an important role in HMGCS2 regulation of the autophagy.

SHMT2 Mediates Small-Molecule-Induced Alleviation of Alzheimer Pathology Via the 5'UTR-dependent ADAM10 Translation Initiation.

It is long been suggested that one-carbon metabolism (OCM) is associated with Alzheimer's disease (AD), whereas the potential mechanisms remain poorly understood. Taking advantage of chemical biology, that mitochondrial serine hydroxymethyltransferase (SHMT2) directly regulated the translation of ADAM metallopeptidase domain 10 (ADAM10), a therapeutic target for AD is reported. That the small-molecule kenpaullone (KEN) promoted ADAM10 translation via the 5' untranslated region (5'UTR) and improved cognitive functions in APP/PS1 mice is found. SHMT2, which is identified as a target gene of KEN and the 5'UTR-interacting RNA binding protein (RBP), mediated KEN-induced ADAM10 translation in vitro and in vivo. SHMT2 controls AD signaling pathways through binding to a large number of RNAs and enhances the 5'UTR activity of ADAM10 by direct interaction with GAGGG motif, whereas this motif affected ribosomal scanning of eukaryotic initiation factor 2 (eIF2) in the 5'UTR. Together, KEN exhibits therapeutic potential for AD by linking OCM with RNA processing, in which the metabolic enzyme SHMT2 "moonlighted" as RBP by binding to GAGGG motif and promoting the 5'UTR-dependent ADAM10 translation initiation.

Cosmosiin Increases ADAM10 Expression via Mechanisms Involving 5'UTR and PI3K Signaling.

The α-secretase "a disintegrin and metalloproteinase domain-containing protein" (ADAM10) is involved in the processing of amyloid precursor protein (APP). Upregulation of ADAM10 precludes the generation of neurotoxic ß-amyloid protein (Aß) and represents a plausible therapeutic strategy for Alzheimer's disease (AD). In this study, we explored compounds that can potentially promote the expression of ADAM10. Therefore, we performed high-throughput small-molecule screening in SH-SY5Y (human neuroblastoma) cells that stably express a luciferase reporter gene driven by the ADAM10 promoter, including a portion of its 5'-untranslated region (5'UTR). This has led to the discovery of cosmosiin (apigenin 7-O-ß-glucoside). Here, we report that in human cell lines (SH-SY5Y and HEK293), cosmosiin proportionally increased the levels of the immature and mature forms of the ADAM10 protein without altering its mRNA level. This effect was attenuated by translation inhibitors or by deleting the 5'UTR of ADAM10, suggesting that a translational mechanism was responsible for the increased levels of ADAM10. Luciferase deletion assays revealed that the first 144 nucleotides of the 5'UTR were necessary for mediating the cosmosiin-induced enhancement of ADAM10 expression in SH-SY5Y cells. Cosmosiin failed to increase the levels of the ADAM10 protein in murine cells, which lack native expression of the ADAM10 transcript containing the identified 5'UTR element. The potential signaling pathway may involve phosphatidylinositide 3-kinase (PI3K) because pharmacological inhibition of PI3K attenuated the effect of cosmosiin on the expression of the ADAM10 protein. Finally, cosmosiin attenuated Aß generation because the levels of Aß40/42 in HEK-APP cells were significantly reduced after cosmosiin treatment. Collectively, we found that the first 144 nucleotides of the ADAM10 5'UTR, and PI3K signaling, are involved in cosmosiin-induced enhancement of the expression of ADAM10 protein. These results suggest that cosmosiin may be a potential therapeutic agent in the treatment of AD.

Phorbol esters dPPA/dPA promote furin expression involving transcription factor CEBPß in neuronal cells.

Using high-throughput small molecule screening targeting furin gene, we identified that phorbol esters dPPA (12-Deoxyphorbol 13-phenylacetate 20-acetate) and dPA (12-Deoxyphorbol 13-acetate) significantly increased furin protein and mRNA expression in SH-SY5Y cells. This effect was prevented by PKC (protein kinase C) inhibitor calphostin C but not Ro318220, suggesting that the C1 domain, rather than the catalytic domain of PKC plays an important role. Luciferase assay revealed that nucleotides -7925 to -7426 were sufficient to mediate dPPA/dPA enhancement of furin P1 promoter activity. RNA interference of transcriptional factors CEBPß (CCAAT/enhancer-binding protein ß) and GATA1 revealed that knockdown of CEBPß significantly attenuated the effect of dPPA on furin expression. Pharmacological inhibition of ERK and PI3K but not TGFß receptor diminished the up-regulation of furin by dPPA. These results suggested that in neuronal cells, transcriptional activation of furin by dPPA/dPA may be initiated by C1 domain containing proteins including PKC; the intracellular signaling involves ERK and PI3K and transcription factor CEBPß.