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Mitochondria are essential for many cellular functions such as oxidative phosphorylation and calcium homeostasis; consequently, mitochondrial dysfunction could cause many diseases, including neurological disorders. Recently, mitochondrial dynamics, such as fusion, fission, and transportation, have been visualized in living cells by using time-lapse imaging systems. The changes in mitochondrial morphology could be an indicator for estimating the activity of mitochondrial biological function. Here, we report a transgenic mouse strain, mtDsRed2-Tg, which expresses a red fluorescent protein, DsRed2, exclusively in mitochondria. Mitochondrial morphology could be clearly observed in various tissues of this strain under confocal microscope. Recently, many transgenic mouse strains in which enhanced green fluorescent protein (EGFP)-tagged proteins of interest are expressed have been established for physiological analysis in vivo. After mating these strains with mtDsRed2-Tg mice, red-colored mitochondria and green-colored proteins were detected simultaneously using fluorescent imaging systems, and the interactions between mitochondria and those proteins could be morphologically analyzed in cells and tissues of the F(1) hybrids. Thus, mtDsRed2-Tg mice can be a powerful tool for bioimaging studies on mitochondrial functions.

PINK1 and Parkin were first identified as the causal genes responsible for familial forms of early-onset Parkinson’s disease (PD), a prevalent neurodegenerative disorder. PINK1 encodes a mitochondrial serine/threonine protein kinase, whereas Parkin encodes an ubiquitin-protein ligase. PINK1 and Parkin cooperate to maintain mitochondrial integrity; however, the detailed molecular mechanism of how Parkin-catalyzed ubiquitylation results in mitochondrial integrity remains an enigma. In this study, we show that Parkin-catalyzed K63-linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin. In addition, p62/SQSTM1 (hereafter referred to as p62) was recruited to depolarized mitochondria after Parkin-directed ubiquitylation. Intriguingly, deletion of p62 in mouse embryonic fibroblasts resulted in a gross loss of mitochondrial perinuclear clustering but did not hinder mitochondrial degradation. Thus, p62 is required for ubiquitylation-dependent clustering of damaged mitochondria, which resembles p62-mediated ‘aggresome’ formation of misfolded/unfolded proteins after ubiquitylation.

Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and lens fiber proteins.