Age-dependent increase of cytoskeletal components in sensory axons in human skin.

Aging is a complex process characterized by several molecular and cellular imbalances. The composition and stability of the neuronal cytoskeleton is essential for the maintenance of homeostasis, especially in long neurites. Using human skin biopsies containing sensory axons from a cohort of healthy individuals, we investigate alterations in cytoskeletal content and sensory axon caliber during aging via quantitative immunostainings. Cytoskeletal components show an increase with aging in both sexes, while elevation in axon diameter is only evident in males. Transcriptomic data from aging males illustrate various patterns in gene expression during aging. Together, the data suggest gender-specific changes during aging in peripheral sensory axons, possibly influencing cytoskeletal functionality and axonal caliber. These changes may cumulatively increase susceptibility of aged individuals to neurodegenerative diseases.

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

BACKGROUND & AIMS: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. METHODS: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. RESULTS: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. CONCLUSION: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. LAY SUMMARY: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Meclizine Preconditioning Protects the Kidney Against Ischemia-Reperfusion Injury.

Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a 'nutrient-sensitized' chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.

ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org.

Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction.

Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K(+) currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.

Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.

Most cells have the inherent capacity to shift their reliance on glycolysis relative to oxidative metabolism, and studies in model systems have shown that targeting such shifts may be useful in treating or preventing a variety of diseases ranging from cancer to ischemic injury. However, we currently have a limited number of mechanistically distinct classes of drugs that alter the relative activities of these two pathways. We screen for such compounds by scoring the ability of >3,500 small molecules to selectively impair growth and viability of human fibroblasts in media containing either galactose or glucose as the sole sugar source. We identify several clinically used drugs never linked to energy metabolism, including the antiemetic meclizine, which attenuates mitochondrial respiration through a mechanism distinct from that of canonical inhibitors. We further show that meclizine pretreatment confers cardioprotection and neuroprotection against ischemia-reperfusion injury in murine models. Nutrient-sensitized screening may provide a useful framework for understanding gene function and drug action within the context of energy metabolism.