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.

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 (TNF␣, IL-1␤) 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.Multiple signaling pathways have been identified that promote growth and survival of neurons and thereby facilitate the formation of synaptic connections that are essential for learning, memory, and the development of the CNS (1-3). Neurotransmitter and neurotrophic receptors, non-receptor tyrosine kinases, and other signaling mediators aggregate to mold and shape postsynaptic densities to permit high-fidelity signal transduction and the regulation of neuronal function (4 -6). A major non-protein component of synapses is cholesterol, which can be a limiting factor in synapse development, synaptic activity, and transmitter release (7).Increasing evidence shows that membrane/lipid rafts, discrete regions of the plasma membrane enriched in cholesterol, glycosphingolipids, and sphingomyelin organize prosurvival and progrowth neuronal signaling pathways (8 -10), regulate cAMP formation (11), and are essential for synapse development, stabilization, and maintenance (7, 12). Caveolin (Cav), 2 a cholesterol binding protein and scaffolding protein found within membrane/lipid rafts (13), organizes and targets certain neuronal growth-promoting proteins, such as components of the neurotransmitter and neurotrophic receptor signaling pathways, to membrane/lipid rafts. These include NMDA receptors, AMPA receptors, Trk receptors, GPC receptors, and Src family kinases (9, 14 -16). These receptors and signaling molecules can enhance cAMP formation, an essential second messenger for promoting neuronal growth and dendritic arborization (17-21), and are found within membrane/lipid rafts in growth cones (6). In the setting of traumatic brain injury and neurodegenerative disorders, interventions that activate signaling pathways to stimulate cAMP production thus have the potential to improve...

cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism, and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrialdependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 genetransferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release, and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart. ischemia/reperfusion | oxidative stress

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved-caspase 9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase 3, 8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its GPI anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

Copper transporter 1 (CTR1) is the major copper (Cu) influx transporter in mammalian cells. We report here that CTR1 is required for the activation of signaling to the MAPK pathway by the ligands of three major receptor tyrosine kinases (RTK) including FGF, PDGF and EGF. Induction of Erk1/2 phosphorylation was compared in isogenic wild type CTR1+/+ and CTR1−/− cells. Whereas all three ligands increased pErk1/2 in the CTR1+/+ cells, they failed to do this in CTR1−/− cells. While FGF did not enhance the phosphorylation of AKT in the CTR1+/+ cells, both PDGF and EGF increased pAKT in the CTR1+/+ but not CTR1−/− cells. The deficit in Erk1/2 phosphorylation in the CTR1−/− cells was rescued by adding Cu to the medium, and it was induced in CTR1+/+ cells by treatment with a Cu chelator. Intracellular Cu availability was reduced in the CTR1−/− cells as reflected by increased expression of the Cu chaperone CCS. The failure of RTK-induced signaling to both Erk1/2 and AKT suggested the presence of a Cu-dependent step upstream of Ras. The Cu-dependent enzyme SOD1 is responsible for generating the hydrogen peroxide in response to RTK activation that serves to inhibit phosphatases that normally limit RTK signaling. SOD1 activity was reduced by a factor of 17-fold in the CTR1−/− cells, and addition of hydrogen peroxide restored signaling. We conclude that Cu acquired from CTR1 is required for signaling in pathways regulated by RTKs that play major roles in development and cancer.

Scope The flavanol (-)-epicatechin (Epi), a component of cacao, has cardiac protective benefits in humans. Our previous study demonstrated Epi has δ-opioid receptor (DOR) binding activity and promotes cardiac protection. Here we examined the effects of 10 days of Epi treatment on: cardiac mitochondrial respiration, ROS production, calcium swelling, and mitochondrial membrane fluidity. Methods & Results Mice were randomized into four groups: (1) Control (Saline), (2) Naltrindole (Nalt; DOR antagonist), (3) Epi, and (4) Epi+Nalt and received 1 mg kg−1 Epi or water via oral gavage. Nalt groups received 5 mg kg−1 ip per day for 10 days. Significant increases in mitochondrial respiration and enhanced free radical production during state 3 respiration were observed with Epi. Additionally, we observed significant increases in rigidity of mitochondrial membranes and resistance to calcium induced mitochondrial swelling with Epi treatment. Blocking the DOR with Nalt resulted in decreases in all of the observed parameters by Epi treatment. Conclusion These findings indicate that Epi induces an integrated response that includes metabolic and structural changes in cardiac mitochondria resulting in greater functional capacity via DOR. Mitochondrial targeted effects of epicatechin may explain the physiologic benefit observed on cardiac protection and support epicatechin’s potential clinical application as a cardiac protective mimetic.

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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 (Cav), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of Caveolin isoforms in microglia physiology is debatable. We propose 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 (CV) 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.