Mitophagy reporter mouse analysis reveals increased mitophagy activity in disuse-induced muscle atrophy.

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

Unique structure of ozoralizumab, a trivalent anti-TNFα NANOBODY® compound, offers the potential advantage of mitigating the risk of immune complex-induced inflammation.

Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.

Ozoralizumab, a Humanized Anti-TNFα NANOBODY® Compound, Exhibits Efficacy Not Only at the Onset of Arthritis in a Human TNF Transgenic Mouse but Also During Secondary Failure of Administration of an Anti-TNFα IgG.

Although the introduction of tumor necrosis factor (TNF) inhibitors represented a significant advance in the treatment of rheumatoid arthritis (RA), traditional anti-TNFα antibodies are somewhat immunogenic, and their use results in the formation of anti-drug antibodies (ADAs) and loss of efficacy (secondary failure). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. In this study we investigated the suppressant effect of ozoralizumab and adalimumab, an anti-TNFα IgG, on arthritis and induction of ADAs in human TNF transgenic mice. Ozoralizumab markedly suppressed arthritis progression and did not induce ADAs during long-term administration. We also developed an animal model of secondary failure by repeatedly administering adalimumab and found that switching from adalimumab to ozoralizumab was followed by superior anti-arthritis efficacy in the secondary-failure animal model. Moreover, ozoralizumab did not form large immune complexes that might lead to ADA formation. The results of our studies suggest that ozoralizumab, which exhibited low immunogenicity in the animal model used and has a different antibody structure from that of IgGs, is a promising candidate for the treatment of RA patients not only at the onset of RA but also during secondary failure of anti-TNFα treatment.

A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis.

In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.

AMSH is required to degrade ubiquitinated proteins in the central nervous system.

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH(-/-) mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH(-/-) mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.

Inhibition of tumor growth and metastasis by depletion of vesicular sorting protein Hrs: its regulatory role on E-cadherin and beta-catenin.

Abnormally high signals from receptor tyrosine kinases (RTK) are associated with carcinogenesis, and impaired deactivation of RTKs may also be a mechanism in cancer. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is one of the master regulators that sort activated receptors toward lysosomes and shut down their signals. Hrs contains a ubiquitin-interacting motif and is involved in the endosomal sorting of monoubiquitinated membrane proteins, such as growth factor receptor and E-cadherin. Here, we investigated the role of Hrs in determining the malignancy of cancer cells and discovered that the targeted disruption of Hrs by small interfering RNA effectively attenuated the proliferation, anchorage-independent growth, tumorigenesis, and metastatic potential of HeLa cells in vitro and in vivo. The restoration of Hrs expression increased cell proliferation and anchorage-independent growth in a mouse embryonic fibroblast line established from a Hrs knockout mouse. Further analysis revealed that Hrs depletion was associated with the up-regulation of E-cadherin and reduced beta-catenin signaling. The aberrant accumulation of E-cadherin most likely resulted from impaired E-cadherin degradation in lysosomes. These results suggest that Hrs may play a critical role in determining the malignancy of cancer cells by regulating the degradation of E-cadherin.

Endosomal sorting complex required for transport proteins in cancer pathogenesis, vesicular transport, and non-endosomal functions.

Endosomal sorting complex required for transport (ESCRT) proteins form a multicomplex sorting machinery that controls multivesicular body (MVB) formation and the sorting of ubiquitinated membrane proteins to the endosomes. Being sorted to the MVB generally results in the lysosome-dependent degradation of cell-surface receptors, and defects in this machinery induce dysregulated receptor traffic and turnover. Recent lessons from gene targeting and silencing methodologies have implicated the ESCRT in normal development, cell differentiation, and growth, as well as in the budding of certain enveloped viruses. Furthermore, it is becoming apparent that the dysregulation of ESCRT proteins is involved in the development of various human diseases, including many types of cancers and neurodegenerative disorders. Here, we summarize the roles of ESCRT proteins in MVB sorting processes and the regulation of tumor cells, and we discuss some of their other functions that are unrelated to vesicular transport.

AMSH, an ESCRT-III associated enzyme, deubiquitinates cargo on MVB/late endosomes.

The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.

Survival of parvovirus B19-infected cells by cellular autophagy.

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.

Hrs, a mammalian master molecule in vesicular transport and protein sorting, suppresses the degradation of ESCRT proteins signal transducing adaptor molecule 1 and 2.

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

Mechanism of p38 MAP kinase activation in vivo.

The p38 mitogen-activated protein kinase (MAPK) is activated in vitro by three different protein kinases: MKK3, MKK4, and MKK6. To examine the relative roles of these protein kinases in the mechanism of p38 MAP kinase activation in vivo, we examined the effect of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway. We show that MKK3 and MKK6are essential for tumor necrosis factor-stimulated p38 MAPK activation. In contrast, ultraviolet radiation-stimulated p38 MAPK activation was mediated by MKK3, MKK4, and MKK6. Loss of p38 MAPK activation in the mutant cells was associated with defects in growth arrest and increased tumorigenesis. These data indicate that p38 MAPK is regulated by the coordinated and selective actions of three different protein kinases in response to cytokines and exposure to environmental stress.