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Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pancaspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy.Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and PARKIN are causal genes for autosomal recessive early-onset parkinsonism (1). PINK1 is a unique Ser/Thr kinase localized on the outer membrane of damaged mitochondria, where it is subsequently autophosphorylated, followed by the formation of a larger protein complex that contains a translocase of the outer membrane (TOM) 4 complex (2-4). PINK1localized on damaged mitochondria selectively recruits PAR-KIN (5, 6), and phosphorylates PARKIN to uncover latent ligase activity (7). PINK1 and phosphorylated PARKIN share a cooperative role to modify mitochondrial outer membrane proteins with phospho-ubiquitin chains, and mitochondria decorated by poly-ubiquitin chains are eliminated by selective mitochondrial autophagy (1), thereby maintaining mitochondrial quality. Programmed cell death serves fundamental functions in tissue development and homeostasis and is associated with several human pathologies, including neurodegradation, autoimmune diseases, and cancer (8). Apoptosis, the best studied form of programmed cell death, is characterized by cell shrinkage, blebbing, nuclear fragmentation, and chromatin condensation, and it requires caspase activation (9). Many studies have revealed caspase-independent but genetically regulated forms of cell death that are classified according to their distinct morphologic features and specific inhibitors (10). PARKIN prevents cells from dying in response to proapoptotic stimuli (11,12). The cytoprotective effects of PARKIN are relatively accepted because loss of PARKIN function leads to pro...

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Age is necessary information for the study of life history of wild animals. A general method to estimate the age of odontocetes is counting dental growth layer groups (GLGs). However, this method is highly invasive as it requires the capture and handling of individuals to collect their teeth. Recently, the development of DNA-based age estimation methods has been actively studied as an alternative to such invasive methods, of which many have used biopsy samples. However, if DNA-based age estimation can be developed from fecal samples, age estimation can be performed without touching or disrupting individuals, thus establishing an entirely non-invasive method. We developed an age estimation model using the methylation rate of two gene regions, GRIA2 and CDKN2A, measured through methylation-sensitive high-resolution melting (MS-HRM) from fecal samples of wild Indo-Pacific bottlenose dolphins (Tursiops aduncus). The age of individuals was known through conducting longitudinal individual identification surveys underwater. Methylation rates were quantified from 36 samples. Both gene regions showed a significant correlation between age and methylation rate. The age estimation model was constructed based on the methylation rates of both genes which achieved sufficient accuracy (after LOOCV: MAE = 5.08, R2 = 0.34) for the ecological studies of the Indo-Pacific bottlenose dolphins, with a lifespan of 40-50 years. This is the first study to report the use of non-invasive fecal samples to estimate the age of marine mammals.

Age is an important parameter that provides a better understanding towards biodemographic trends–development, survival, reproduction and environmental effects–which is critical for conservation. However, current methods to estimate age is difficult in many species and no standardised technique has been adopted. Here, we focus on the endangered Asian elephants (Elephas maximus) to examine the potential of estimating age from DNA methylation through two candidate age-related epigenetic genes and develop an age estimation model. DNA was extracted from blood samples (n = 53) from 25 known-aged captive individuals. Methylation rates of two genetic regions: RALYL and TET2 were measured via methylation-sensitive high-resolution melting (MS-HRM) which is a labour-, time-, and cost-effective method. The developed DNA methylation-based age estimation model showed a significant correlation with chronological age for both markers: RALYL (cor = 0.54, p < 0.001) and TET2(cor = −0.60, p < 0.001). The final age estimation model combining both genes showed a mean absolute deviation (MAD) of 5.56 years. This study highlights MS-HRM as a practical and convenient method to investigate the relationship between epigenetic modifications in age-related genes and chronological age in Asian elephants, and its potential to provide key life history information for future implications.