Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].

Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Statins, which reduce LDL-cholesterol by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin-induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity-dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin-1, dystrophin, epidermal growth factor receptor, and insulin receptor-ß; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte-equivalent HL-1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long-term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size-variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria- and endoplasmic reticulum-related genes; and a 21% increase in mortality in cardiac-specific vinculin-knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long-term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.-Godoy, J. C., Niesman, I. R., Busija, A. R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D. M., Kararigas, G., Patel, H. H., Zemljic-Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Neuron-Targeted Caveolin-1 Promotes Ultrastructural and Functional Hippocampal Synaptic Plasticity.

A delicate interneuronal communication between pre- and postsynaptic membranes is critical for synaptic plasticity and the formation of memory. Evidence shows that membrane/lipid rafts (MLRs), plasma membrane microdomains enriched in cholesterol and sphingolipids, organize presynaptic proteins and postsynaptic receptors necessary for synaptic formation and signaling. MLRs establish a cell polarity that facilitates transduction of extracellular cues to the intracellular environment. Here we show that neuron-targeted overexpression of an MLR protein, caveolin-1 (SynCav1), in the adult mouse hippocampus increased the number of presynaptic vesicles per bouton, total excitatory type I glutamatergic synapses, number of same-dendrite multiple-synapse boutons, increased myelination, increased long-term potentiation, and increased MLR-localized N-methyl-d-aspartate receptor subunits (GluN1, GluN2A, and GluN2B). Immunogold electron microscopy revealed that Cav-1 localizes to both the pre- and postsynaptic membrane regions as well as in the synaptic cleft. These findings, which are consistent with a significant increase in ultrastructural and functional synaptic plasticity, provide a fundamental framework that underlies previously demonstrated improvements in learning and memory in adult and aged mice by SynCav1. Such observations suggest that Cav-1 and MLRs alter basic aspects of synapse biology that could serve as potential therapeutic targets to promote neuroplasticity and combat neurodegeneration in a number of neurological disorders.

Pathophysiology Associated with Traumatic Brain Injury: Current Treatments and Potential Novel Therapeutics.

Traumatic brain injury (TBI) is one of the leading causes of death of young people in the developed world. In the United States alone, 1.7 million traumatic events occur annually accounting for 50,000 deaths. The etiology of TBI includes traffic accidents, falls, gunshot wounds, sports, and combat-related events. TBI severity ranges from mild to severe. TBI can induce subtle changes in molecular signaling, alterations in cellular structure and function, and/or primary tissue injury, such as contusion, hemorrhage, and diffuse axonal injury. TBI results in blood-brain barrier (BBB) damage and leakage, which allows for increased extravasation of immune cells (i.e., increased neuroinflammation). BBB dysfunction and impaired homeostasis contribute to secondary injury that occurs from hours to days to months after the initial trauma. This delayed nature of the secondary injury suggests a potential therapeutic window. The focus of this article is on the (1) pathophysiology of TBI and (2) potential therapies that include biologics (stem cells, gene therapy, peptides), pharmacological (anti-inflammatory, antiepileptic, progrowth), and noninvasive (exercise, transcranial magnetic stimulation). In final, the review briefly discusses membrane/lipid rafts (MLR) and the MLR-associated protein caveolin (Cav). Interventions that increase Cav-1, MLR formation, and MLR recruitment of growth-promoting signaling components may augment the efficacy of pharmacologic agents or already existing endogenous neurotransmitters and neurotrophins that converge upon progrowth signaling cascades resulting in improved neuronal function after injury.

Caveolin modulates integrin function and mechanical activation in the cardiomyocyte.

ß1 integrins (ß1) transduce mechanical signals in many cells, including cardiac myocytes (CM). Given their close localization, as well as their role in mechanotransduction and signaling, we hypothesized that caveolin (Cav) proteins might regulate integrins in the CM. ß1 localization, complex formation, activation state, and signaling were analyzed using wild-type, Cav3 knockout, and Cav3 CM-specific transgenic heart and myocyte samples. Studies were performed under basal and mechanically loaded conditions. We found that: (1) ß1 and Cav3 colocalize in CM and coimmunoprecipitate from CM protein lysates; (2) ß1 is detected in a subset of caveolae; (3) loss of Cav3 caused reduction of ß1D integrin isoform and active ß1 integrin from the buoyant domains in the heart; (4) increased expression of myocyte Cav3 correlates with increased active ß1 integrin in adult CM; (5) in vivo pressure overload of the wild-type heart results in increased activated integrin in buoyant membrane domains along with increased association between active integrin and Cav3; and (6) Cav3-deficient myocytes have perturbed basal and stretch mediated signaling responses. Thus, Cav3 protein can modify integrin function and mechanotransduction in the CM and intact heart.

Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1.

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-ß-cyclodextrin (MßCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MßCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MßCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 µM MßCD, whereas SLP was more robust and only inhibited with ≥200 µM MßCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice.

BACKGROUND: Traumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI. METHODS: Controlled cortical impact (CCI) model of TBI (3 m/second; 1.0 mm depth; parietal cortex) was performed on wild-type (WT; C57Bl/6), Cav-1 KO, and Cav-3 KO mice. Histology and immunofluorescence microscopy (lesion volume, glia activation), behavioral tests (open field, balance beam, wire grip, T-maze), electrophysiology, electron paramagnetic resonance, membrane fractionation, and multiplex assays were performed. Data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni's multiple comparison. RESULTS: CCI increased cortical and hippocampal injury and decreased expression of MLR-localized synaptic proteins (24 hours), enhanced NADPH oxidase (Nox) activity (24 hours and 1 week), enhanced polysynaptic responses (1 week), and caused hippocampal-dependent learning deficits (3 months). CCI increased brain lesion volume in both Cav-3 and Cav-1 KO mice after 24 hours (P < 0.0001, n = 4; one-way ANOVA). Multiplex array revealed a significant increase in expression of IL-1ß, IL-9, IL-10, KC (keratinocyte chemoattractant), and monocyte chemoattractant protein 1 (MCP-1) in ipsilateral hemisphere and IL-9, IL-10, IL-17, and macrophage inflammatory protein 1 alpha (MIP-1α) in contralateral hemisphere of WT mice after 4 hours. CCI increased IL-2, IL-6, KC and MCP-1 in ipsilateral and IL-6, IL-9, IL-17 and KC in contralateral hemispheres in Cav-1 KO and increased all 10 cytokines/chemokines in both hemispheres except for IL-17 (ipsilateral) and MIP-1α (contralateral) in Cav-3 KO (versus WT CCI). Cav-3 KO CCI showed increased IL-1ß, IL-9, KC, MCP-1, MIP-1α, and granulocyte-macrophage colony-stimulating factor in ipsilateral and IL-1ß, IL-2, IL-9, IL-10, and IL-17 in contralateral hemispheres (P = 0.0005, n = 6; two-way ANOVA) compared to Cav-1 KO CCI. CONCLUSION: CCI caused astrocyte and microglial activation and hippocampal neuronal injury. Cav-1 and Cav-3 KO exhibited enhanced lesion volume and cytokine/chemokine production after CCI. These findings suggest that Cav isoforms may regulate neuroinflammatory responses and neuroprotection following TBI.

Cardioprotective trafficking of caveolin to mitochondria is Gi-protein dependent.

BACKGROUND: Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria. METHODS: Mice were exposed to isoflurane or oxygen vehicle (30 min, ± 36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed. RESULTS: Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P < 0.05]; [caveolin-3: 1.68 ± 0.29 vs. 2.67 ± 0.46; P < 0.05]) and mitochondrial caveolin levels (n = 16 per group; [caveolin-1: 0.87 ± 0.18 vs. 1.89 ± .19; P < 0.05]; [caveolin-3: 1.10 ± 0.29 vs. 2.26 ± 0.28; P < 0.05]), and caveolin-enriched mitochondria exhibited improved respiratory function (n = 4 per group; [state 3/complex I: 10.67 ± 1.54 vs. 37.6 ± 7.34; P < 0.05]; [state 3/complex II: 37.19 ± 4.61 vs. 71.48 ± 15.28; P < 0.05]). Isoflurane increased phosphorylation of survival kinases (n = 8 per group; [protein kinase B: 0.63 ± 0.20 vs. 1.47 ± 0.18; P < 0.05]; [glycogen synthase kinase 3ß: 1.23 ± 0.20 vs. 2.35 ± 0.20; P < 0.05]). The beneficial effects were blocked by pertussis toxin. CONCLUSIONS: Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

Caveolin isoform switching as a molecular, structural, and metabolic regulator of microglia.

Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cavs), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of caveolin isoforms in microglia physiology is debatable. We propose that caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CVs) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.

A Hollow TFG Condensate Spatially Compartmentalizes the Early Secretory Pathway.

In the early secretory pathway, endoplasmic reticulum (ER) and Golgi membranes form a nearly spherical interface. In this ribosome-excluding zone, bidirectional transport of cargo coincides with a spatial segregation of anterograde and retrograde carriers by an unknown mechanism. We show that at physiological conditions, Trk-fused gene (TFG) self-organizes to form a hollow, anisotropic condensate that matches the dimensions of the ER-Golgi interface. Regularly spaced hydrophobic residues in TFG control the condensation mechanism and result in a porous condensate surface. We find that TFG condensates act as a molecular sieve, enabling molecules corresponding to the size of anterograde coats (COPII) to access the condensate interior while restricting retrograde coats (COPI). We propose that a hollow TFG condensate structures the ER-Golgi interface to create a diffusion-limited space for bidirectional transport. We further propose that TFG condensates optimize membrane flux by insulating secretory carriers in their lumen from retrograde carriers outside TFG cages.

Mitochondria-localized caveolin in adaptation to cellular stress and injury.

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae-mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav-3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress-induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav-3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin-knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav-2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria-caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.

Meditation-induced bloodborne factors as an adjuvant treatment to COVID-19 disease.

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. Management of the pandemic has relied mainly on SARS-CoV-2 vaccines, while alternative approaches such as meditation, shown to improve immunity, have been largely unexplored. Here, we probe the relationship between meditation and COVID-19 disease and directly test the impact of meditation on the induction of a blood environment that modulates viral infection. We found a significant inverse correlation between length of meditation practice and SARS-CoV-2 infection as well as accelerated resolution of symptomology of those infected. A meditation "dosing" effect was also observed. In cultured human lung cells, blood from experienced meditators induced factors that prevented entry of pseudotyped viruses for SARS-CoV-2 spike protein of both the wild-type Wuhan-1 virus and the Delta variant. We identified and validated SERPINA5, a serine protease inhibitor, as one possible protein factor in the blood of meditators that is necessary and sufficient for limiting pseudovirus entry into cells. In summary, we conclude that meditation can enhance resiliency to viral infection and may serve as a possible adjuvant therapy in the management of the COVID-19 pandemic.

Neuron-targeted caveolin-1 protein enhances signaling and promotes arborization of primary neurons.

Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1ß) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative brain.

Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation.

BACKGROUND: Propofol exposure to neurons during synaptogenesis results in apoptosis, leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75(NTR)) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75(NTR). METHODS: Days in vitro 5-7 neurons were exposed to propofol (3 µM) for 6 h and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75(NTR-/-) mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 µM, 15 min), a specific p75(NTR) inhibitor. P75(NTR-/-) neurons were transfected for 72 h with a lentiviral vector containing the synapsin-driven p75(NTR) gene (Syn-p75(NTR)) or control vector (Syn-green fluorescent protein) before propofol. To confirm our in vitro findings, wild-type mice and p75(NTR-/-) mice (PND5) were pretreated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. RESULTS: Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75(NTR-/-) neurons. In p75(NTR-/-) neurons transfected with Syn-p75(NTR), propofol significantly increased Cl-Csp3 in comparison with Syn-green fluorescent protein-transfected p75(NTR-/-) neurons. Wild-type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75(NTR-/-) mice. CONCLUSION: These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75(NTR) and the downstream effector RhoA kinase.

Draft Genome Sequence of Nereida sp. Strain MMG025, Isolated from Giant Kelp.

Here, we report the draft genome sequence of Nereida sp. strain MMG025, isolated from the surface of giant kelp and assembled and analyzed by undergraduate students participating in a marine microbial genomics course. A genomic comparison suggests that MMG025 is a novel species, providing a resource for future microbiology and biotechnology investigations.

Loss of the inactive myotubularin-related phosphatase Mtmr13 leads to a Charcot-Marie-Tooth 4B2-like peripheral neuropathy in mice.

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by slowed nerve conduction velocity, axon loss, and distinctive myelin outfolding and infolding. CMT4B is caused by recessive mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P(2), two phosphoinositides that regulate endosomal-lysosomal membrane traffic. Interestingly, nearly half of the metazoan myotubularins are predicted to be catalytically inactive. Both active and inactive myotubularins have essential functions in mammals and in Caenorhabditis elegans. MTMR2 and MTMR13 are active and inactive PI 3-phosphatases, respectively, and the two proteins have been shown to directly associate, although the functional significance of this association is not well understood. To establish a mouse model of CMT4B2, we disrupted the Mtmr13 gene. Mtmr13-deficient mice develop a peripheral neuropathy characterized by reduced nerve conduction velocity and myelin outfoldings and infoldings. Dysmyelination is evident in Mtmr13-deficient nerves at 14 days and worsens throughout life. Thus, loss of Mtmr13 in mice leads to a peripheral neuropathy with many of the key features of CMT4B2. Although myelin outfoldings and infoldings occur most frequently at the paranode, our morphological analyses indicate that the ultrastructure of the node of Ranvier and paranode is intact in Mtmr13-deficient nerve fibers. We also found that Mtmr2 levels are decreased by approximately 50% in Mtmr13-deficient sciatic nerves, suggesting a mode of Mtmr2 regulation. Mtmr13-deficient mice will be an essential tool for studying how the loss of MTMR13 leads to CMT4B2.

Structural membrane changes induced by pulsed blue light on methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: In a recent study we showed that blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA) by perturbing, depolarizing, and disrupting its cell membrane. PURPOSE: The current study presents visual evidence that the observed biochemical changes also result in cell metabolic changes and structural alteration of the cell membrane. METHODS: Cultures of MRSA were treated with 450 nm pulsed blue light (PBL) at 3 mW/cm2 irradiance, using a sub lethal dose of 2.7 J/cm2 radiant exposure three times at 30-min intervals. Following 24 h incubation at 37 °C, irradiated colonies and control non-irradiated colonies were processed for light and transmission electron microscopy. RESULTS: The images obtained revealed three major effects of PBL; (1) disruption of MRSA cell membrane, (2) alteration of membrane structure, and (3) disruption of cell replication. CONCLUSION: These signs of bacterial inactivation at a dose deliberately selected to be sub-lethal supports our previous finding that rapid depolarization of bacterial cell membrane and disruption of cellular function comprise another mechanism underlying photo-inactivation of bacteria. Further, it affirms the potency of PBL.

Role of caveolin-3 and glucose transporter-4 in isoflurane-induced delayed cardiac protection.

BACKGROUND: Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. The authors tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolae or caveolin dependent. METHODS: An in vivo mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (APC) was tested in wild-type, caveolin-1 knockout, and caveolin-3 knockout mice. Mice were exposed to 30 min of oxygen or isoflurane and allowed to recover for 24 h. After 24 h recovery, mice underwent 30-min coronary artery occlusion followed by 2 h of reperfusion at which time infarct size was determined. Biochemical assays were also performed in excised hearts. RESULTS: Infarct size as a percent of the area at risk was reduced by isoflurane in wild-type (24.0 +/- 8.8% vs. 45.1 +/- 10.1%) and caveolin-1 knockout mice (27.2 +/- 12.5%). Caveolin-3 knockout mice did not show delayed APC (41.5 +/- 5.0%). Microscopically distinct caveolae were observed in wild-type and caveolin-1 knockout mice but not in caveolin-3 knockout mice. Delayed APC increased the amount of caveolin-3 protein but not caveolin-1 protein in discontinuous sucrose-gradient buoyant fractions. In addition, glucose transporter-4 was increased in buoyant fractions, and caveolin-3/glucose transporter-4 colocalization was observed in wild-type and caveolin-1 knockout mice after APC. CONCLUSIONS: These results show that delayed APC involves translocation of caveolin-3 and glucose transporter-4 to caveolae, resulting in delayed protection in the myocardium.