MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

Mitochondrial dynamics define muscle fiber type by modulating cellular metabolic pathways.

Skeletal muscle is highly developed after birth, consisting of glycolytic fast-twitch and oxidative slow-twitch fibers; however, the mechanisms of fiber-type-specific differentiation are poorly understood. Here, we found an unexpected role of mitochondrial fission in the differentiation of fast-twitch oxidative fibers. Depletion of the mitochondrial fission factor dynamin-related protein 1 (Drp1) in mouse skeletal muscle and cultured myotubes results in specific reduction of fast-twitch muscle fibers independent of respiratory function. Altered mitochondrial fission causes activation of the Akt/mammalian target of rapamycin (mTOR) pathway via mitochondrial accumulation of mTOR complex 2 (mTORC2), and rapamycin administration rescues the reduction of fast-twitch fibers in vivo and in vitro. Under Akt/mTOR activation, the mitochondria-related cytokine growth differentiation factor 15 is upregulated, which represses fast-twitch fiber differentiation. Our findings reveal a crucial role of mitochondrial dynamics in the activation of mTORC2 on mitochondria, resulting in the differentiation of muscle fibers.

Effects of imeglimin on mitochondrial function, AMPK activity, and gene expression in hepatocytes.

Imeglimin is a recently launched antidiabetic drug structurally related to metformin. To provide insight into the pharmacological properties of imeglimin, we investigated its effects on hepatocytes and compared them with those of metformin. The effects of imeglimin on mitochondrial function in HepG2 cells or mouse primary hepatocytes were examined with an extracellular flux analyzer and on gene expression in HepG2 cells by comprehensive RNA-sequencing analysis. The effects of the drug on AMPK activity in HepG2 cells, mouse primary hepatocytes, and mouse liver were also examined. Treatment of HepG2 cells or mouse primary hepatocytes with imeglimin reduced the oxygen consumption rate coupled to ATP production. Imeglimin activated AMPK in these cells whereas the potency was smaller than metformin. Bolus administration of imeglimin in mice also activated AMPK in the liver. Whereas the effects of imeglimin and metformin on gene expression in HepG2 cells were similar overall, the expression of genes encoding proteins of mitochondrial respiratory complex III and complex I was upregulated by imeglimin but not by metformin. Our results suggest that imeglimin and metformin exert similar pharmacological effects on mitochondrial respiration, AMPK activity, and gene expression in cultured hepatocytes, whereas the two drugs differ in their effects on the expression of certain genes related to mitochondrial function.

Pearson syndrome-like anemia induced by accumulation of mutant mtDNA and anemia with imbalanced white blood cell lineages induced by Drp1 deletion in a murine model.

Regulation of mitochondrial respiration and morphology is important for maintaining steady-state hematopoiesis, yet few studies have comparatively evaluated the effects of abnormal mitochondrial respiration and dynamics on blood-cell differentiation in isolation or combination. This study sought to explore these effects in mouse models with one or both of the following deficits: a large-scale deletion of mitochondrial DNA (ΔmtDNA), accumulated to varying extents, or knockout of the mitochondrial fission factor Drp1. Each deficit was found to independently provoke anemia but with clearly different manifestations. The former showed signs of aberrant respiration, analogous to Pearson syndrome, while the latter showed signs of abnormal mitochondrial dynamics and was associated with changes in the relative proportions of leukocyte lineages. Combining these deficits acted to amplify abnormal iron metabolism in erythropoiesis, exacerbating anemia in an additive manner. Our results indicate that mitochondrial respiration and dynamics play distinct roles in different sets of processes and cell lineages in hematopoietic differentiation.

Aclarubicin, an anthracycline anti-cancer drug, fluorescently contrasts mitochondria and reduces the oxygen consumption rate in living human cells.

Aclarubicin (Acla), an effective anthracycline chemotherapeutic agent for hematologic cancers and solid tumors, is documented to perturb chromatin function via histone eviction and DNA topoisomerase inhibition in the nucleus, but much less attention has been paid to cytotoxic function in the cytoplasm. Here, we showed that Acla emitted fluorescence and that human cervical cancer HeLa cells exposed to Acla exhibited bright fluorescence signals in the cytoplasm when fluorescence microscopy was performed using the red filter (excitation 530-550nm/emission 575nm). Intriguingly, most of the signals appeared to be partitioned and enriched in entangled tubule-like structures; moreover, these signals merged with the mitochondria-specific MitoTracker signals. Notably, analysis of mitochondrial respiratory activity revealed that the oxygen consumption rate was decreased in Acla-treated cells. These findings suggest that Acla accumulates efficiently in the mitochondria of living human cells and leads to mitochondrial dysfunction, implying a previously overlooked cytotoxicity of Acla in the cytoplasm and adding mechanistic insight of the anti-cancer activity, as well as the side effects, of Acla/anthracycline-based chemotherapy.

HECT-type ubiquitin ligase ITCH targets lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) and prevents LAPTM5-mediated cell death.

LAPTM5 (lysosomal-associated protein multispanning transmembrane 5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. Although the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is regulated negatively by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death.

ITCH is a putative target for a novel 20q11.22 amplification detected in anaplastic thyroid carcinoma cells by array-based comparative genomic hybridization.

Anaplastic thyroid carcinoma (ATC) is one of the most virulent of all human malignancies, with a mean survival time among patients of less than 1 year after diagnosis. To date, however, cytogenetic information on this disease has been very limited. During the course of a program to screen a panel of ATC cell lines for genomic copy-number aberrations using array-based comparative genomic hybridization, we identified a high-level amplification of the ITCH gene, which is mapped to 20q11.22 and belongs to the homologous to the E6-associated protein carboxylterminus ubiquitin ligase family. The expression of ITCH was increased in 4 of 14 ATC cell lines (28.6%), including 8305C in which there was a copy-number amplification of this gene, and six of seven primary cases (85.7%). Among the primary thyroid tumors, a considerable number of ITCH high expressers was found in ATC (40/45, 88.9%), papillary thyroid carcinoma (25/25, 100%), and papillary microcarcinoma (25/25, 100%). Furthermore, knockdown of ITCH by specific small interfering RNA significantly inhibited the growth of ITCH-overexpressing cells, whereas ectopic overexpression of ITCH promoted growth of ATC cell lines with relatively weak expression. These observations indicate ITCH to be the most likely target for 20q11.22 amplification and to play a crucial role in the progression of thyroid carcinoma.