MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes

Late-onset megaconial myopathy in mice lacking group I Paks

Abstract Background Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. Methods We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation. Results dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. Conclusions Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes