Presequence protease reverses mitochondria-specific amyloid-ß-induced mitophagy to protect mitochondria.

Amyloid-ß (Aß) peptide is accumulated in the mitochondria and has been shown to play a central role in the development of Alzheimer's disease (AD). It has been shown that exposure of neurons to aggregated Aß can result in damaged mitochondria and dysregulated mitophagy, indicating that changes in the Aß content of mitochondria may affect the levels of mitophagy and interfere with the progression of AD. However, the direct influence of mitochondrial Aß on mitophagy has not been elucidated. In the present study, the effect of the mitochondria-specific Aß was assessed following a direct change of Aß content in the mitochondria. We directly change mitochondrial Aß by transfecting cells with mitochondria-associated plasmids, including the mitochondrial outer membrane protein translocase 22 (TOMM22) and 40 (TOMM40) or presequence protease (PreP) overexpression plasmids. The changes in the levels of mitophagy were assessed by TEM, Western blot, mito-Keima construct, organelle tracker, and probe JC-1 assay. We demonstrated that increased mitochondrial Aß content enhance mitophagy levels; overexpression of PreP could reverse the mitochondrial Aß-induced mitophagy levels in vivo and in vitro by reversing the levels of reactive oxygen species (ROS) and the mitochondrial membrane potential. The data provide novel insight into the role of mitochondria-specific Aß in the progression of AD pathophysiology.

Depletion of TMEM65 leads to oxidative stress, apoptosis, induction of mitochondrial unfolded protein response, and upregulation of mitochondrial protein import receptor TOMM22.

Mutation in the transmembrane protein 65 gene (TMEM65) results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype. However, neither the function of TMEM65 nor the cellular responses to its depletion have been fully elucidated. Hence, we knocked down TMEM65 in human cultured cells and analyzed the resulting cellular responses. Depletion of TMEM65 led to a mild increase in ROS generation and upregulation of the mRNA levels of oxidative stress suppressors, such as NFE2L2 and SESN3, indicating that TMEM65 knockdown induced an oxidative stress response. A mild induction of apoptosis was also observed upon depletion of TMEM65. Depletion of TMEM65 upregulated protein levels of the mitochondrial chaperone HSPD1 and mitochondrial protease LONP1, indicating that mitochondrial unfolded protein response (UPRmt) was induced in response to TMEM65 depletion. Additionally, we found that the mitochondrial protein import receptor TOMM22 and HSPA9 (mitochondrial Hsp70), were also upregulated in TMEM65-depleted cells. Notably, the depletion of TMEM65 did not lead to upregulation of TOMM22 in an ATF5-dependent manner, although upregulation of LONP1 reportedly occurs in an ATF5-dependent manner. Taken together, our findings suggest that depletion of TMEM65 causes mild oxidative stress and apoptosis, induces UPRmt, and upregulates protein expression of mitochondrial protein import receptor TOMM22 in an ATF5-independent manner.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

Epstein-Barr Virus EBER Transcripts Affect miRNA-Mediated Regulation of Specific Targets and Are Processed to Small RNA Species.

The oncogenic Epstein-Barr virus (EBV) expresses 44 mature microRNAs and two non-coding EBER RNAs of 167 (EBER1) and 172 (EBER2) nt length. MiRNA profiling of NK/T cell lines and primary cells and Northern blotting of EBV-infected cell lines and primary tumors revealed processing of EBER1 to short 5'-derived RNAs of approximately 23, 52 and 70 nt (EBER123, EBER152, and EBER170) and of EBER2 to 3' fragments. The biogenesis of these species is independent of Dicer, and EBER123 does not act like a miRNA OPEN ACCESS Non-Coding RNA 2015, 1 171 to target its complementary sequence. EBER1, EBER2 and EBER123 were bound by the lupus antigen (La), a nuclear and cytoplasmic protein that facilitates RNAi. Consistent with this, the EBERs affect regulation of interleukin 1alpha (IL1α) and RAC1 reporters harboring miR target sequences, targets of miR-142-3p. However, the EBERs have no effect upon another target of miR-142-3p, ADCY9, nor on TOMM22, a target of ebv-miR-BART16, indicative of selective modulation of gene expression by the EBERs.