Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia.

Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin ß, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.

Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization.

Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.

A new radiographic view of the hindfoot.

BACKGROUND: A new radiographic view was proposed to evaluate the coronal plane alignment of the hindfoot under weightbearing conditions. METHODS: We studied 46 feet of individuals with normal appearing asymptomatic feet. On the antero-posterior roentgenogram using this new method, the line from the top of the sustentaculum tali to the lateral-inferior end of the posterior articular surface of the talus was obtained as the standard line showing varus or valgus of the calcaneus. We defined the angle between the longitudinal axis of the tibia and the standard line as described above as Varus-Valgus angle (V-V angle). RESULTS: The mean (±SD) V-V angle of the 46 feet studied was 76.4 (±3.6) degrees. CONCLUSION: The findings from this study indicate that it is possible to estimate the alignment of the hindfoot quantitatively by comparing individuals to the mean V-V angle that we calculated in our sample, which was 76.4 degrees.

Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP.

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human alpha(1)-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.

Static and dynamic biomechanical properties of the regenerating rabbit Achilles tendon.

BACKGROUND: Since tendons show viscoelastic behavior, dynamic viscoelastic properties should be assessed in addition to static biomechanical properties. We evaluated differences between static and dynamic biomechanical properties of the regenerating rabbit Achilles tendon following tenotomy. METHODS: At 3, 6, or 12 weeks after right Achilles tenotomy, the right (regenerating) and left (control) tendons were collected with the calcaneus from 49 rabbits. A unidirectional failure test and a dynamic viscoelastic test were conducted. FINDINGS: Tensile strength and Young's modulus (static biomechanical properties) in the regenerating group at Week 6 were significantly greater than at Week 3, while at Week 12, these were significantly greater than at Week 6. However, even at Week 12, both parameters were less than in the control group. The value of tan delta represents dynamic viscoelasticity, a smaller tan delta indicates greater elasticity. tan delta for the regenerating group was significantly greater than for the control group at Week 3, but regenerating and control groups did not significantly differ at Week 6. No marked change was seen from Weeks 6 to 12 in the regenerating group, and no significant difference in tan delta was evident between the regenerating and control groups at Week 12. INTERPRETATION: Dynamic biomechanical properties of regenerating rabbit Achilles tendons may improve more rapidly than static biomechanical properties. Ability to tolerate dynamic movement in the healing Achilles tendon may improve more rapidly than ability to withstand static stresses.

Simultaneous induction of the four subunits of the TRAP complex by ER stress accelerates ER degradation.

The mammalian translocon-associated protein (TRAP) complex comprises four transmembrane protein subunits in the endoplasmic reticulum. The complex associates with the Sec61 translocon, although its function in vivo remains unknown. Here, we show the involvement of the TRAP complex in endoplasmic reticulum-associated degradation (ERAD). All four subunits are induced simultaneously by endoplasmic reticulum stresses from the X-box-binding protein 1/inositol-requiring 1alpha pathway. RNA interference knockdown of each subunit causes disruption of the native complex and significant delay in the degradation of various ERAD substrates, including the alpha1-antitrypsin null Hong Kong variant (NHK). In a pulse-chase experiment, the TRAP complex associated with NHK at a late stage, indicating its involvement in the ERAD pathway rather than in biosynthesis of nascent polypeptides in the endoplasmic reticulum. In addition, the TRAP complex bound preferentially to misfolded proteins rather than correctly folded wild-type substrates. Thus, the TRAP complex induced by the unfolded protein response pathway might discriminate ERAD substrates from correctly folded substrates, accelerating degradation.

Utility of grommets for implant arthroplasty of the great toe in a patient with rheumatoid arthritis: a case report.

We report a case of rheumatoid arthritis treated by bilateral flexible hinge toe implant arthroplasty, using grommets only on one side, which resulted in bilateral fractures requiring removal of the implants 6.5 years after the surgery. Both implants were completely fractured at the bottom of the distal stems. Macroscopically, synovitis was present around both fractured stems, although the severity of synovitis and fragmentation of the fractured implant was relatively mild on the right side in which grommets were used. The shape of the body of the fractured implant was relatively preserved on the right side in which grommets were used. There was no damage or fracture of the grommets. The grommet might have acted to prevent pressures and scratches that would cause synovitis and deformity of the body of the implant, but might not completely prevent fractures of implants.