A role for BCL2L13 and autophagy in germline purifying selection of mtDNA.

Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNAAla gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a statistically robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.

Ironing the mitochondria: Relevance to its dynamics.

The mitochondrion is "jack of many trades and master of one". Despite being a master in energy generation, it plays a significant role in other cellular processes, including calcium homeostasis, cell death, and iron metabolism. Since mitochondria employ the majority of cellular iron, it plays a central role in the iron homeostasis. Iron could be a major regulator of mitochondrial dynamics as the excess of iron leads to oxidative stress, which causes a disturbance in mitochondrial dynamics. Remarkably, abnormal iron accumulation has been observed in the brain regions of the neurodegenerative disorders patients. These neurodegenerative disorders are also often associated with the abnormal mitochondrial dynamics. Here in this article, we will mainly discuss the studies focused on unravelling the role of iron in mitochondrial dynamics.

Correction to: Interdependence of laforin and malin proteins for their stability and functions could underlie the molecular basis of locus heterogeneity in Lafora disease.

Correction to: J. Biosci. 40(5), December 2015, 863-871 https://doi.org/10.1007/s12038-015-9570-0 The image of anti-Myc blot of figure 2C (third panel; Malin-Myc [C26S]) was inadvertently used once again for the c-tubulin loading control of figure 2B. The revised figure 2B with the correct image of the c-tubulin loading control is given below. The interpretation and conclusion provided in the article do not change because of the correction.

Loss of laforin or malin results in increased Drp1 level and concomitant mitochondrial fragmentation in Lafora disease mouse models.

Lafora disease (LD) is an autosomal recessive form of a fatal disorder characterized by the myoclonus epilepsy, ataxia, psychosis, dementia, and dysarthria. A hallmark of LD is the presence of abnormal glycogen inclusions called Lafora bodies in the affected tissues including the neurons. LD can be caused by defects either in the laforin phosphatase coded by the EPM2A gene or in the malin E3 ubiquitin ligase coded by the NHLRC1 gene. The mouse models of LD, created by the targeted disruption of the LD genes, display several neurodegenerative changes. Prominent among them are the autophagic defects, abnormally large lysosomes, neurofibrillary tangles, amyloid beta deposits, and abnormal mitochondria. However, whether or not such neurodegenerative changes are a direct effect of the loss of laforin/malin was not unequivocally established. Here, we show that laforin- or malin-deficient neurons and fibroblasts display a significantly higher number of fragmented mitochondria. Loss of laforin or malin resulted in increased levels of the mitochondrial fission GTPase Drp1, its enhanced mitochondrial targeting, and increased intracellular calcium levels. Intriguingly, laforin and malin display opposite effects on the cellular level of parkin, an ubiquitin ligase of Drp1; loss of laforin led to reduced levels of parkin while the loss of malin resulted in increased parkin levels. Laforin and malin, however, interact with and positively regulate the activity of parkin, thus explaining the molecular basis of increased Drp1 levels in LD tissues. Our results suggest that laforin and malin are novel regulators of mitochondrial quality control pathway and that the mitochondrial dysfunction resulting from the increased Drp1 levels could underlie neuropathology in LD.

Heat shock modulates the subcellular localization, stability, and activity of HIPK2.

The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress - such as hypoxia, oxidative stress, or UV damage - is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

Interdependence of laforin and malin proteins for their stability and functions could underlie the molecular basis of locus heterogeneity in Lafora disease.

Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the EPM2A gene coding for the laforin protein phosphatase or the NHLRC1 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each other's function. Using cell biological assays we demonstrate here that (i) malin promotes its own degradation via autoubiquitination, (ii) laforin prevents the auto-degradation of malin by presenting itself as a substrate and (iii) malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each other's stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneity observed in LD.

Lafora disease proteins laforin and malin negatively regulate the HIPK2-p53 cell death pathway.

Lafora disease (LD) is an autosomal recessive, progressive, and fatal form of a neurodegenerative disorder characterized by the presence of Lafora polyglucosan bodies. LD is caused by defects in either the laforin protein phosphatase or the malin E3 ubiquitin ligase. Laforin and malin were shown play key roles in proteolytic processes, unfolded stress response, and glycogen metabolism. Therefore, the LD proteins laforin and malin are thought to function as pro-survival factors and their loss thus could result in neurodegeneration. To understand the molecular pathway leading to the cell death in LD, in the present study, we investigated the possible role of LD proteins in the p53-mediated cell death pathway. We show that loss of laforin or malin results in the increased level and activity of p53, both in cellular and animal models of LD, and that this is primarily due to the increased levels of Hipk2, a proapoptotic activator of p53. Overexpression of laforin or malin confers protection against Hipk2-mediated cell death by targeting the Hipk2 to the cytoplasmic compartment. Taken together, our study strengthens the notion that laforin and malin are pro-survival factors, and that the activation of Hipk2-p53 cell death pathway might underlie neurodegeneration in LD.

Malin and laforin are essential components of a protein complex that protects cells from thermal stress.

The heat-shock response is a conserved cellular process characterized by the induction of a unique group of proteins known as heat-shock proteins. One of the primary triggers for this response, at least in mammals, is heat-shock factor 1 (HSF1)--a transcription factor that activates the transcription of heat-shock genes and confers protection against stress-induced cell death. In the present study, we investigated the role of the phosphatase laforin and the ubiquitin ligase malin in the HSF1-mediated heat-shock response. Laforin and malin are defective in Lafora disease (LD), a neurodegenerative disorder associated with epileptic seizures. Using cellular models, we demonstrate that these two proteins, as a functional complex with the co-chaperone CHIP, translocate to the nucleus upon heat shock and that all the three members of this complex are required for full protection against heat-shock-induced cell death. We show further that laforin and malin interact with HSF1 and contribute to its activation during stress by an unknown mechanism. HSF1 is also required for the heat-induced nuclear translocation of laforin and malin. This study demonstrates that laforin and malin are key regulators of HSF1 and that defects in the HSF1-mediated stress response pathway might underlie some of the pathological symptoms in LD.