Nicotine inhalant via E-cigarette facilitates sensorimotor function recovery by upregulating neuronal BDNF-TrkB signalling in traumatic brain injury.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) causes lifelong physical and psychological dysfunction in affected individuals. The current study investigated the effects of chronic nicotine exposure via E-cigarettes (E-cig) (vaping) on TBI-associated behavioural and biochemical changes. EXPERIMENTAL APPROACH: Adult C57/BL6J male mice were subjected to controlled cortical impact (CCI) followed by daily exposure to E-cig vapour for 6 weeks. Sensorimotor functions, locomotion, and sociability were subsequently evaluated by nesting, open field, and social approach tests, respectively. Immunoblots were conducted to examine the expression of mature brain-derived neurotrophic factor (mBDNF) and associated downstream proteins (p-Erk, p-Akt). Histological analyses were performed to evaluate neuronal survival and neuroinflammation. KEY RESULTS: Post-injury chronic nicotine exposure significantly improved nesting performance in CCI mice. Histological analysis revealed increased survival of cortical neurons in the perilesion cortex with chronic nicotine exposure. Immunoblots revealed that chronic nicotine exposure significantly up-regulated mBDNF, p-Erk and p-Akt expression in the perilesion cortex of CCI mice. Immunofluorescence microscopy indicated that elevated mBDNF and p-Akt expression were mainly localized within cortical neurons. Immunolabelling of Iba1 demonstrated that chronic nicotine exposure attenuated microglia-mediated neuroinflammation. CONCLUSIONS AND IMPLICATIONS: Post-injury chronic nicotine exposure via vaping facilitates recovery of sensorimotor function by upregulating neuroprotective mBDNF/TrkB/Akt/Erk signalling. These findings suggest potential neuroprotective properties of nicotine despite its highly addictive nature. Thus, understanding the multifaceted effects of chronic nicotine exposure on TBI-associated symptoms is crucial for paving the way for informed and properly managed therapeutic interventions.

Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Age-Dependent Behavioral and Metabolic Assessment of App NL-G-F/NL-G-F Knock-in (KI) Mice.

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models which were widely used in the past decades share a common limitation: The overexpression of APP and overproduction of amyloid-beta (Aß) are accompanied by other APP peptide fragments, which could introduce artificial and non-clinically relevant phenotypes. Here, we performed an in-depth and time-resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations (App NL-G-F/NL-G-F ), termed APP knock-in (APPKI) mice. Our result showed that APPKI mice exhibited fear learning deficits at 6-m age and contextual memory deficit at 12-m age. Histopathological analysis revealed mild amyloidosis (6E10) accompanied by microgliosis (Iba1) as early as 3 months, which progressed significantly together with significant astrocytosis at 6 and 12 m. We further analyzed hippocampal mitochondrial dysfunction by multiple assays, while 3-m APPKI mice brain mitochondrial function remains a similar level as WT mice. Significant mitochondrial dysfunction characterized by decreased ATP production and higher membrane potential with subsequent overproduction of reactive oxygen species (ROS) was observed in mitochondria isolated from 7-m APPKI mice hippocampal tissue. Morphologically, these mitochondria were larger in volume with a decreased level of mitochondrial fusion protein mitofusin-2 (MFN2). At 12 months, APPKI mice exhibit a significantly decreased total mitochondrial oxygen consumption rate (OCR) in isolated hippocampal mitochondria detected by high-resolution respirometry. These data indicate early mitochondrial dysfunction in the brain at pre-symptomatic age in the App NL-G-F/NL-G-mice, which may play a key role in the progression of the disease. Moreover, the identified behavioral and bioenergetic alterations in this clinically relevant AD mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD.

Synapsin-Promoted Caveolin-1 Overexpression Maintains Mitochondrial Morphology and Function in PSAPP Alzheimer's Disease Mice.

Mitochondrial dysfunction plays a pivotal role in the Alzheimer's Disease (AD) pathology. Disrupted mitochondrial dynamics (i.e., fusion/fission balance), which are essential for normal mitochondria structure and function, are documented in AD. Caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein regulates metabolic pathways in several different cell types such as hepatocytes and cancer cells. Previously, we have shown decreased expression of Cav-1 in the hippocampus of 9-month (m) old PSAPP mice, while hippocampal overexpression of neuron-targeted Cav-1 using the synapsin promoter (i.e., SynCav1) preserved cognitive function, neuronal morphology, and synaptic ultrastructure in 9 and 12 m PSAPP mice. Considering the central role of energy production in maintaining normal neuronal and synaptic function and survival, the present study reveals that PSAPP mice exhibit disrupted mitochondrial distribution, morphometry, and respiration. In contrast, SynCav1 mitigates mitochondrial damage and loss and enhances mitochondrial respiration. Furthermore, by examining mitochondrial dynamics, we found that PSAPP mice showed a significant increase in the phosphorylation of mitochondrial dynamin-related GTPase protein (DRP1), resulting in excessive mitochondria fragmentation and dysfunction. In contrast, hippocampal delivery of SynCav1 significantly decreased p-DRP1 and augmented the level of the mitochondrial fusion protein, mitofusin1 (Mfn1) in PSAPP mice, a molecular event, which may mechanistically explain for the preserved balance of mitochondria fission/fusion and metabolic resilience in 12 m PSAPP-SynCav1 mice. Our data demonstrate the critical role for Cav-1 in maintaining normal mitochondrial morphology and function through affecting mitochondrial dynamics and explain a molecular and cellular mechanism underlying the previously reported neuroprotective and cognitive preservation induced by SynCav1 in PSAPP mouse model of AD.

Synapsin-caveolin-1 gene therapy preserves neuronal and synaptic morphology and prevents neurodegeneration in a mouse model of AD.

Alzheimer's disease (AD) is the most common form of neurodegeneration and cognitive dysfunction in the elderly. Identifying molecular signals that mitigate and reverse neurodegeneration in AD may be exploited therapeutically. Transgenic AD mice (PSAPP) exhibit learning and memory deficits at 9 and 11 months, respectively, with associated decreased expression of caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein necessary for synaptic and neuroplasticity. Neuronal-targeted gene therapy using synapsin-Cav-1 cDNA (SynCav1) was delivered to the hippocampus of PSAPP mice at 3 months using adeno-associated virus serotype 9 (AAV9). Bilateral SynCav1 gene therapy was able to preserve MLRs profile, learning and memory, hippocampal dendritic arbor, synaptic ultrastructure, and axonal myelin content in 9- and 11-month PSAPP mice, independent of reducing toxic amyloid deposits and astrogliosis. Our data indicate that SynCav1 gene therapy may be an option for AD and potentially in other forms of neurodegeneration of unknown etiology.

Preclinical validation of a potent γ-secretase modulator for Alzheimer's disease prevention.

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer's disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aß42 and to a lesser extent Aß40, while concomitantly augmenting production of Aß38 and Aß37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aß42 in rat brain by 50%.

Cathepsin B in neurodegeneration of Alzheimer's disease, traumatic brain injury, and related brain disorders.

Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.

Depletion of Caveolin-1 in Type 2 Diabetes Model Induces Alzheimer's Disease Pathology Precursors.

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of late-onset Alzheimer's disease (AD). However, the mechanism underlying the development of late-onset AD is largely unknown. Here we show that levels of the endothelial-enriched protein caveolin-1 (Cav-1) are reduced in the brains of T2DM patients compared with healthy aging, and inversely correlated with levels of ß-amyloid (Aß). Depletion of Cav-1 is recapitulated in the brains of db/db (Leprdb ) diabetic mice and corresponds with recognition memory deficits as well as the upregulation of amyloid precursor protein (APP), BACE-1, a trending increase in ß-amyloid Aß42/40 ratio and hyperphosphorylated tau (p-tau) species. Importantly, we show that restoration of Cav-1 levels in the brains of male db/db mice using adenovirus overexpressing Cav-1 (AAV-Cav-1) rescues learning and memory deficits and reduces pathology (i.e., APP, BACE-1 and p-tau levels). Knocking down Cav-1 using shRNA in HEK cells expressing the familial AD-linked APPswe mutant variant upregulates APP, APP carboxyl terminal fragments, and Aß levels. In turn, rescue of Cav-1 levels restores APP metabolism. Together, these results suggest that Cav-1 regulates APP metabolism, and that depletion of Cav-1 in T2DM promotes the amyloidogenic processing of APP and hyperphosphorylation of tau. This may suggest that depletion of Cav-1 in T2DM underlies, at least in part, the development of AD and imply that restoration of Cav-1 may be a therapeutic target for diabetic-associated sporadic AD.SIGNIFICANCE STATEMENT More than 95% of the Alzheimer's patients have the sporadic late-onset form (LOAD). The cause for late-onset Alzheimer's disease is unknown. Patients with Type 2 diabetes mellitus have considerably higher incidence of cognitive decline and AD compared with the general population, suggesting a common mechanism. Here we show that the expression of caveolin-1 (Cav-1) is reduced in the brain in Type 2 diabetes mellitus. In turn, reduced Cav-1 levels induce AD-associated neuropathology and learning and memory deficits. Restoration of Cav-1 levels rescues these deficits. This study unravels signals underlying LOAD and suggests that restoration of Cav-1 may be an effective therapeutic target.

Interactions between estrogen receptors and metabotropic glutamate receptors and their impact on drug addiction in females.

Contribution to Special Issue on Fast effects of steroids. Estrogen receptors α and ß (ERα and ERß) have a unique relationship with metabotropic glutamate receptors (mGluRs) in the female rodent brain such that estradiol is able to recruit intracellular G-protein signaling cascades to influence neuronal physiology, structure, and ultimately behavior. While this association between ERs and mGluRs exists in many cell types and brain regions, its effects are perhaps most striking in the nucleus accumbens (NAc). This review will discuss the original characterization of ER/mGluR signaling and how estradiol activity in the NAc confers increased sensitivity to drugs of abuse in females through this mechanism.

Neuroadaptations in the dentate gyrus following contextual cued reinstatement of methamphetamine seeking.

Abstinence from unregulated methamphetamine self-administration increases hippocampal dependent, context-driven reinstatement of methamphetamine seeking. The current study tested the hypothesis that alterations in the functional properties of granule cell neurons (GCNs) in the dentate gyrus (DG) of the hippocampus in concert with altered expression of synaptic plasticity-related proteins and ultrastructural changes in the DG are associated with enhanced context-driven methamphetamine-seeking behavior. Whole-cell patch-clamp recordings were performed in acute brain slices from methamphetamine naïve (controls) and methamphetamine experienced animals (during acute withdrawal, during abstinence, after extinction and after reinstatement). Spontaneous excitatory postsynaptic currents (sEPSCs) and intrinsic excitability were recorded from GCNs. Reinstatement of methamphetamine seeking increased sEPSC frequency and produced larger amplitude responses in GCNs compared to controls and all other groups. Reinstatement of methamphetamine seeking reduced spiking capability in GCNs compared to controls, and all other groups, as indicated by reduced intrinsic spiking elicited by increasing current injections, membrane resistance and fast after hyperpolarization. In rats that reinstated methamphetamine seeking, these altered electrophysiological properties of GCNs were associated with enhanced expression of Fos, GluN2A subunits and PSD95 and reduced expression of GABAA subunits in the DG and enhanced expression of synaptic PSD in the molecular layer. The alterations in functional properties of GCNs and plasticity related proteins in the DG paralleled with no changes in structure of microglial cells in the DG. Taken together, our results demonstrate that enhanced reinstatement of methamphetamine seeking results in alterations in intrinsic spiking and spontaneous glutamatergic synaptic transmission in the GCNs and concomitant increases in neuronal activation of GCNs, and expression of GluNs and decreases in GABAA subunits that may contribute to the altered synaptic connectivity-neuronal circuitry-and activity in the hippocampus, and enhance propensity for relapse.

Multifunctional stimuli responsive polymer-gated iron and gold-embedded silica nano golf balls: Nanoshuttles for targeted on-demand theranostics.

Multi-functional nanoshuttles for remotely targeted and on-demand delivery of therapeutic molecules and imaging to defined tissues and organs hold great potentials in personalized medicine, including precise early diagnosis, efficient prevention and therapy without toxicity. Yet, in spite of 25 years of research, there are still no such shuttles available. To this end, we have designed magnetic and gold nanoparticles (NP)-embedded silica nanoshuttles (MGNSs) with nanopores on their surface. Fluorescently labeled Doxorubicin (DOX), a cancer drug, was loaded in the MGNSs as a payload. DOX loaded MGNSs were encapsulated in heat and pH sensitive polymer P(NIPAM-co-MAA) to enable controlled release of the payload. Magnetically-guided transport of MGNSs was examined in: (a) a glass capillary tube to simulate their delivery via blood vessels; and (b) porous hydrogels to simulate their transport in composite human tissues, including bone, cartilage, tendon, muscles and blood-brain barrier (BBB). The viscoelastic properties of hydrogels were examined by atomic force microscopy (AFM). Cellular uptake of DOX-loaded MGNSs and the subsequent pH and temperature-mediated release were demonstrated in differentiated human neurons derived from induced pluripotent stem cells (iPSCs) as well as epithelial HeLa cells. The presence of embedded iron and gold NPs in silica shells and polymer-coating are supported by SEM and TEM. Fluorescence spectroscopy and microscopy documented DOX loading in the MGNSs. Time-dependent transport of MGNSs guided by an external magnetic field was observed in both glass capillary tubes and in the porous hydrogel. AFM results affirmed that the stiffness of the hydrogels model the rigidity range from soft tissues to bone. pH and temperature-dependent drug release analysis showed stimuli responsive and gradual drug release. Cells' viability MTT assays showed that MGNSs are non-toxic. The cell death from on-demand DOX release was observed in both neurons and epithelial cells even though the drug release efficiency was higher in neurons. Therefore, development of smart nanoshuttles have significant translational potential for controlled delivery of theranostics' payloads and precisely guided transport in specified tissues and organs (for example, bone, cartilage, tendon, bone marrow, heart, lung, liver, kidney, and brain) for highly efficient personalized medicine applications.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

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 glycophosphatidylinositol 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.

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.

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

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.

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.

Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity.

BACKGROUND: Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8(-/-)), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system. METHODOLOGY/PRINCIPAL FINDINGS: Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8(-/-) mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging. CONCLUSIONS: Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma.

Volatile anesthetics protect cancer cells against tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via caveolins.

BACKGROUND: Volatile anesthetics have a dual effect on cell survival dependent on caveolin expression. The effect of volatile anesthetics on cancer cell survival and death after anesthetic exposure has not been well investigated. The authors examined the effects of isoflurane exposure on apoptosis and its regulation by caveolin-1 (Cav-1). METHODS: The authors exposed human colon cancer cell lines to isoflurane and proapoptotic stimuli and assessed what role Cav-1 plays in cell protection. They evaluated apoptosis using assays for nucleosomal fragmentation, cleaved caspase 3 expression, and caspase activity assays. To test the mechanism, they used pharmacologic inhibitors (i.e., pertussis toxin) and assessed changes in glycolysis. RESULTS: Apoptosis as measured by nucleosomal fragmentation was enhanced by isoflurane (1.2% in air) in HT29 (by 64% relative to control, P < 0.001) and decreased in HCT116 (by 23% relative to control, P < 0.001) cells. Knockdown of Cav-1 in HCT116 cells increased the sensitivity to apoptotic stimuli but not with scrambled small interfering RNA (siRNA) treatment (19.7 ± 0.4 vs. 20.0 ± 0.6, P = 0.7786 and 19.7 ± 0.5 vs. 16.3 ± 0.4, P = 0.0012, isoflurane vs. control in Cav-1 small interfering RNA vs. scrambled small interfering RNA treated cells, respectively). The protective effect of isoflurane with various exposure times on apoptosis was enhanced in HT29 cells overexpressing Cav-1 (P < 0.001 by two-way ANOVA). Pertussis toxin effectively blocked the antiapoptotic effect of isoflurane exhibited by Cav-1 in all cell lines. Cav-1 cells had increased glycolysis with isoflurane exposure; however, in the presence of tumor necrosis factor-related apoptosis-inducing ligand, this increase in glycolysis was maintained in HT29-Cav-1 but not control cells. CONCLUSION: Brief isoflurane exposure leads to resistance against apoptosis via a Cav-1-dependent mechanism.

Cardiac-specific overexpression of caveolin-3 attenuates cardiac hypertrophy and increases natriuretic peptide expression and signaling.

OBJECTIVES: We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy. BACKGROUND: Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization. METHODS: We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes. RESULTS: The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-ß-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression. CONCLUSIONS: These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.