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Axonal degeneration occurs in multiple neurodegenerative disorders of the central and peripheral nervous system. Although the under-lying molecular pathways leading to axonal degeneration are incompletely understood, accumulating evidence suggests contributions of impaired mitochondrial function, disrupted axonal transport, and/or dysfunctional intracellular Ca2+-homeostasis in the injurious cascade associated with axonal degeneration. Utilizing an in vitro model of axonal degeneration, we studied a subset of mouse peripheral sensory neurons in which neurites were exposed selectively to conditions associated with the pathogenesis of axonal neuropathies in vivo. Rotenone-induced mitochondrial dysfunction resulted in neurite degeneration accompanied by reduced ATP levels and increased ROS levels in neurites. Blockade of voltage-gated sodium channels with TTX and reverse (Ca2+-importing) mode of the sodium-calcium exchanger (NCX) with KB-R7943 partially protected rotenone-treated neurites from degeneration, suggesting a contribution of sodium channels and reverse NCX activity to the degeneration of neurites resulting from impaired mitochondrial function. Pharmacological inhibition of the Na+/K+ -ATPase with ouabain induced neurite degeneration, which was attenuated by TTX and KB-R7943, supporting a contribution of sodium channels in axonal degenerative pathways accompanying impaired Na+/K+-ATPase activity. Conversely, oxidant stress (H2O2 )-induced neurite degeneration was not attenuated by TTX. Our results demonstrate that both energetic and oxida-tive stress targeted selectively to neurites induces neurite degeneration and that blockade of sodium channels and of reverse NCX activity blockade partially protects neurites from injury due to energetic stress, but not from oxidative stress induced by H2O2. [ABSTRACT FROM AUTHOR]

AbstractDamaged and dysfunctional mitochondria are proposed to be removed by autophagy. However, selective degradation of damaged mitochondria by autophagy (mitophagy) has yet to be experimentally verified. In this study, we investigated the cellular fate of individual mitochondria damaged by photoirradiation in hepatocytes isolated from transgenic mice expressing green fluorescent protein fused to microtubule-associated protein 1 light chain 3, a marker of forming and newly formed autophagosomes. Photoirradiation with 488-nm light induced mitochondrial depolarization (release of tetramethylrhodamine methylester [TMRM]) in a dose-dependent fashion. At lower doses of light, mitochondria depolarized transiently with re-polarization within 3 min. With greater light, mitochondrial depolarization became irreversible. Irreversible, but not reversible, photodamage induced autophagosome formation after 32±5 min. Photodamage-induced mitophagy was independent of TMRM, as photodamage also induced mitophagy in the absence of TMRM. Photoirradiation with 543-nm light did not induce mitophagy. As revealed by uptake of LysoTracker Red, mitochondria weakly acidified after photodamage before a much stronger acidification after autophagosome formation. Photodamage-induced mitophagy was not blocked by phosphatidylinositol 3-kinase inhibition with 3-methyladenine (10 mM) or wortmannin (100 nM). In conclusion, individual damaged mitochondria become selectively degraded by mitophagy, but photodamage-induced mitophagic sequestration occurs independently of the phosphatidylinositol 3-kinase signaling pathway, the classical upstream signaling pathway of nutrient deprivation-induced autophagy. Antioxid. Redox Signal.14, 1919–1928. [ABSTRACT FROM AUTHOR]

Examines the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-messenger RNA polyuridine tract and 3' splice-site sequences. Significance of the chemical-shift perturbations; Identification of a characteristic alipathic residue of the RNA-binding domain of sex-lethal.

Autophagy rids cells of unwanted organelles and recovers nutrients during nutrient deprivation. Microtubule-associated protein 1 light chain 3 (LC3) is recruited to autophagosomes. Here, we study the dynamics of mitochondrial autophagy (mitophagy) in transgenic GFP-LC3 mouse hepatocytes co-loaded with tetramethylrhodamine methylester (TMRM), Lysotracker Red (LTR) or/and PicoGreen (Pico). During nutrient deprivation in Krebs-Ringer HEPES buffer with glucagon (KRH/G), GFP-LC3 (green) rings formed as small green dots decreased. The dots near mitochondria grew into crescents that sequestered TMRM-labeled mitochondria into GFP-LC3 rings in 6 to 12 min. GFP-LC3 sequestered entire mitochondria (42.4%) or portions taken after fission from the ends (43.1%) or middle (9.1%) At completion of sequestration, mitochondria depolarized (loss of TMRM). GFP-LC3 rings acidified (took up LTR) after another 5 min. Pico-labeled mtDNA became entrapped within LTR-labeled autophagosomes and disappeared during lysosomal digestion. In conclusion, LC3-containing membranes envelope all or parts of individual mitochondria during nutrient deprivation-induced mitophagy. Mitochondria then depolarize, which is followed by autophagosomal acidification and digestion. Mitophagy appears to be an important mechanism for mtDNA turnover. [ABSTRACT FROM AUTHOR]