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).

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.

Relations of combat stress and posttraumatic stress disorder to 24-h plasma and cerebrospinal fluid interleukin-6 levels and circadian rhythmicity.

BACKGROUND: Acute and chronic stress can lead to a dysregulation of the immune response. Growing evidence suggests peripheral immune dysregulation and low-grade systemic inflammation in posttraumatic stress disorder (PTSD), with numerous reports of elevated plasma interleukin-6 (IL-6) levels. However, only a few studies have assessed IL-6 levels in the cerebrospinal fluid (CSF). Most of those have used single time-point measurements, and thus cannot take circadian level variability and CSF-plasma IL-6 correlations into account. METHODS: This study used time-matched, sequential 24-h plasma and CSF measurements to investigate the effects of combat stress and PTSD on physiologic levels and biorhythmicity of IL-6 in 35 male study volunteers, divided in 3 groups: (PTSD = 12, combat controls, CC = 12, and non-deployed healthy controls, HC = 11). RESULTS: Our findings show no differences in diurnal mean concentrations of plasma and CSF IL-6 across the three comparison groups. However, a significantly blunted circadian rhythm of plasma IL-6 across 24 h was observed in all combat-zone deployed participants, with or without PTSD, in comparison to HC. CSF IL-6 rhythmicity was unaffected by combat deployment or PTSD. CONCLUSIONS: Although no significant group differences in mean IL-6 concentration in either CSF or plasma over a 24-h timeframe was observed, we provide first evidence for a disrupted peripheral IL-6 circadian rhythm as a sequel of combat deployment, with this disruption occurring in both PTSD and CC groups. The plasma IL-6 circadian blunting remains to be replicated and its cause elucidated in future research.

Posttraumatic stress disorder influences the nociceptive and intrathecal cytokine response to a painful stimulus in combat veterans.

OBJECTIVE: Although posttraumatic stress disorder (PTSD) and chronic pain frequently occur in tandem, the pathophysiological mechanisms mediating this comorbidity are poorly understood. Because excessive inflammation occurs in both conditions, we examined the cerebrospinal fluid (CSF) concentrations of inflammatory response mediators interleukin 1-beta (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-alpha (TNFα) and interleukin 10 (IL-10) after prolonged suprathreshold pain stimulus in 21 male combat veterans; 10 with PTSD and 11 combat controls (CC). METHODS: After completing baseline quantitative sensory testing (QST) and psychological profiling, all patients received an injection of capsaicin into the quadriceps muscle. Spontaneously reported pain was measured for 30min after the capsaicin injection. The evoked pain measure of temporal summation was tested between 70 and 110min post capsaicin injection. Inflammatory (IL-1ß, IL-6, IL-8 TNFα) and anti-inflammatory (IL-10) CSF cytokines were measured before (baseline) and after capsaicin injection over a time frame of 110min. RESULTS: Following intramuscular capsaicin injection, pro-inflammatory cytokines [TNFα, IL-6, IL-8] significantly increased (percent rise from baseline) in both groups, whereas IL-1ß significantly increased in the PTSD group only. The anti-inflammatory cytokine IL-10 showed an immediate (within 10min) increase in the CC group; however, the IL-10 increase in the PTSD group was delayed and not consistently elevated until 70min post injection. CONCLUSION: These findings show significant central nervous system (CNS) differences in the inflammatory response to a deep pain stimulus in combat veterans with and without PTSD. They support the concept that abnormally elevated neuroinflammatory response to pain stimuli may be one CNS mechanism accounting for the high co-occurrence of PTSD and pain.

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.

Circadian rhythmicity, variability and correlation of interleukin-6 levels in plasma and cerebrospinal fluid of healthy men.

BACKGROUND: Interleukin-6 (IL-6) is a cytokine with pleiotropic actions in both the periphery of the body and the central nervous system (CNS). Altered IL-6 secretion has been associated with inflammatory dysregulation and several adverse health consequences. However, little is known about the physiological circadian characteristics and dynamic inter-correlation between circulating and CNS IL-6 levels in humans, or their significance. METHODS: Simultaneous assessment of plasma and cerebrospinal fluid (CSF) IL-6 levels was performed hourly in 11 healthy male volunteers over 24h, to characterize physiological IL-6 secretion levels in both compartments. RESULTS: IL-6 levels showed considerable within- and between-subject variability in both plasma and CSF, with plasma/CSF ratios revealing consistently higher levels in the CSF. Both CSF and plasma IL-6 levels showed a distinctive circadian variation, with CSF IL-6 levels exhibiting a main 24h, and plasma a biphasic 12h, circadian component. Plasma peaks were roughly at 4 p.m. and 4 a.m., while the CSF peak was at around 7 p.m. There was no correlation between coincident CSF and plasma IL-6 values, but evidence for significant correlations at a negative 7-8h time lag. CONCLUSIONS: This study provides evidence in humans for a circadian IL-6 rhythm in CSF and confirms prior observations reporting a plasma biphasic circadian pattern. Our results indicate differential IL-6 regulation across the two compartments and are consistent with local production of IL-6 in the CNS. Possible physiological significance is discussed and implications for further research are highlighted.

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.

An observational study of the influence of "white-coat hypertension" on day-of-surgery blood pressure determinations.

BACKGROUND: Because decisions as to what range of intraoperative blood pressure (BP) is consistent with cerebral well-being are often made in reference to "baseline BP," we sought to determine whether day-of-surgery BPs accurately reflect baseline BP, as defined by ambulatory clinic BPs over the preceding 7 months. METHODS: Consecutive patients (n=101) who were severely hypertensive (Severe-HTN), systolic (S)>160 mm Hg, or diastolic (D)>100 at first operating room BP (1st OR-BP) were identified retrospectively. Two additional groups were formed from patients whose 1st OR-BP was moderately hypertensive (Mod-HTN, systolic BP=140 to 159 and/or diastolic BP=90 to 99; and normotensive, SBP=110 to 139 and DBP<89). 1st OR-BP was compared with: (1) BP before transfer to the OR (Pre-OR-BP); (2) BP during ambulatory evaluation 1 to 30 days preoperatively (Preop-Eval-BP); and (3) Baseline-BP (average of at least 3 ambulatory clinic BPs during the preceding 7 months). Comorbidity data were collected. RESULTS: For Severe-HTNs, 1st OR-BP, and Pre-OR-BP (expressed as mean arterial pressure) exceeded Baseline-BP by 16.4±11.6 (SD) and 5.2±11.6 (SD), respectively (P<0.05). Preop-Eval-BP was not different from Baseline-BP. For Mod-HTNs, 1st OR-BP exceeded Baseline-BP by 7.4±8.1 (SD) (P<0.05). But, Pre-OR-BP and Preop-Eval-BP did not differ from Baseline-BP. Among normotensives, 1st OR-BP was not different from Preop-Eval-BP or Baseline-BP. Hypertension, number of antihypertensive medications, vascular diagnoses (peripheral, coronary, cerebral), diabetes, and renal disease were more common in the hypertensive groups. The number of antihypertensive medications, a history of coronary disease, and insulin administration were predictors of an increase in 1st OR-BP over Baseline-BP. CONCLUSIONS: For most patients whose 1st OR-BP is hypertensive, that BP is greater than ambulatory clinic BPs recorded during the preceding 7 months. For most patients with Severe-HTN at 1st OR-BP, day-of-surgery BPs overestimate Baseline-BP and reference to prehospitalization BPs is advisable. When 1st OR-BP is normotensive, that BP usually reflects Baseline-BP.

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.

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.

Role of decoy molecules in neuronal ischemic preconditioning.

AIMS: Decoy receptors bind with TNF related apoptosis inducing ligands (TRAIL) but do not contain the cytoplasmic domains necessary to transduce apoptotic signals. We hypothesized that decoy receptors may confer neuronal protection against lethal ischemia after ischemic preconditioning (IPC). MAIN METHOD: Mixed cortical neurons were exposed to IPC one day prior to TRAIL treatment or lethal ischemia. KEY FINDINGS: IPC increased decoy receptor but reduced death receptor expression compared to lethal ischemia. IPC-induced increase in decoy receptor expression was reduced by prior treatment with CAPE, a nuclear factor-kappa B inhibitor (NFκB). SIGNIFICANCE: Expression of decoy molecules, dependent on NFκB, may mediate neuronal survival induced by IPC.

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning.

BACKGROUND: Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte-specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. METHODS AND RESULTS: Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte-specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3beta phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3beta and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. CONCLUSIONS: Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Caveolin-1 expression is essential for N-methyl-D-aspartate receptor-mediated Src and extracellular signal-regulated kinase 1/2 activation and protection of primary neurons from ischemic cell death.

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activation and downstream signaling are important for neuronal function. Activation of prosurvival Src family kinases and extracellular signal-regulated kinase (ERK) 1/2 is initiated by NMDAR activation, but the cellular organization of these kinases in relation to NMDARs is not entirely clear. We hypothesized that caveolin-1 scaffolds and coordinates protein complexes involved in NMDAR signaling and that this organization is necessary for neuronal preconditioning, whereby NMDAR activation protects neurons from subsequent ischemic cell death. We found that sublethal ischemia (SLI) or preconditioning via NMDA treatment of primary cortical neurons from neonatal rats or mice increases expression of phosphorylated (P) caveolin-1, P-Src, and P-ERK1/2. The NMDAR antagonist, MK801, or the Src inhibitor, PP2, attenuated SLI-induced preconditioning. NMDAR2B distributed to buoyant fractions and heavy fractions, partially colocalized with caveolin-1 and the membrane raft marker, cholera toxin B. Cultures of primary neurons treated with caveolin-1 small interfering RNA or from caveolin-1(-/-) mice lacked the NMDA-mediated increase in P-Src and P-ERK, as well as SLI- and NMDA-induced preconditioning. Adenovirally mediated expression of caveolin-1 in neurons from caveolin-1(-/-) mice restored NMDA-mediated enhancement of P-Src and P-ERK1/2, redistributed NMDAR2B to buoyant fractions, and enhanced NMDAR2B localization to membrane rafts. We conclude that caveolin-1, perhaps via its ability to scaffold key signaling components, is essential for NMDAR localization to neuronal membrane rafts, NMDAR/Src tyrosine kinase family/ERK signaling, and protection of neurons from ischemic injury and cell death.

Ischemic preconditioning in the brain.

PURPOSE OF REVIEW: Brain ischemia is responsible for significant morbidity and mortality associated with cardiovascular surgery, and is the end result of multiple disease states, including cardiac arrest, stroke, and traumatic brain injury. Despite significant resources dedicated to developing neuroprotective strategies, little progress has been made in this regard. Neuronal ischemic preconditioning is an endogenous neuroprotective strategy that provides sustained and robust ischemic tolerance. Identification of the mechanisms responsible for mediating the preconditioning response may offer novel therapeutic targets and further our understanding of the natural adaptations to brain injury. RECENT FINDINGS: Recent research efforts have elucidated many intracellular signaling pathways that ultimately lead to ischemic tolerance after a preconditioning stimulus. Most of these are associated with glutamate receptor signal transduction, the intracellular kinases, and several transcription regulators. Microarray analysis has identified several gene families that warrant further investigation to identify novel candidates for neuroprotective therapies. These include genes involved in synaptic architecture and signal propagation, cell cycle and transcription regulators, and mediators of apoptosis such as the heat shock proteins and anti-apoptotic mitochondrial proteins. SUMMARY: Neuronal ischemic preconditioning is an endogenous mechanism that leads to robust neuroprotection from ischemia. Identification of the upstream pathways that initiate preconditioning and candidate genes that mediate this phenomenon may offer novel therapeutic targets, with applicability to a variety of disease states and perioperative complications.