R. Anthony DeFazio

Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε

In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of ε protein kinase C (εPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of εPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal εPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of εPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific εPKC activating peptide, tat-ψεRACK (tat-ψε-receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that εPKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal εPKC. Activation of εPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, εPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the εPKC signaling pathway.

ɛPKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.

Differential volatile signatures from skin, naevi and melanoma: a novel approach to detect a pathological process.

Background Early detection of melanoma is of great importance to reduce mortality. Discovering new melanoma biomarkers would improve early detection and diagnosis. Here, we present a novel approach to detect volatile compounds from skin. Methods and Findings We used Head Space Solid Phase Micro-Extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS) to identify volatile signatures from melanoma, naevi and skin samples. We hypothesized that the metabolic state of tissue alters the profile of volatile compounds. Volatiles released from fresh biopsy tissue of melanoma and benign naevus were compared based on their difference in frequency distribution and their expression level. We also analyzed volatile profiles from frozen tissue, including skin and melanoma. Conclusions Three volatiles, 4-methyl decane, dodecane and undecane were preferentially expressed in both fresh and frozen melanoma, indicating that they are candidate biomarkers. Twelve candidate biomarkers evaluated by fuzzy logic analysis of frozen samples distinguished melanoma from skin with 89% sensitivity and 90% specificity. Our results demonstrate proof-of-principle that there is differential expression of volatiles in melanoma. Our volatile metabolomic approach will lead to a better understanding of melanoma and can enable development of new diagnostic and treatment strategies based on altered metabolism.

Calcium Current Subtypes in GnRH Neurons

Abstract Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% ± 2%. The remaining HVA calcium current passed through N-type (27% ± 3%), P-type (15% ± 1%), Q-type (18% ± 3%), and R-type (15% ± 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4–10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.

GABA synapses mediate neuroprotection after ischemic and εPKC preconditioning in rat hippocampal slice cultures

Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the ε-isoform of protein kinase C (εPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and εPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or εPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or ψεRACK, a selective εPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABAA receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, εPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABAA receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9±6.1%. When applied during test ischemia, 100nmol/L bicuculline abolished neuroprotection conferred by either IPC or εPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.

Voltage-Gated Potassium Currents Are Targets of Diurnal Changes in Estradiol Feedback Regulation and Kisspeptin Action on Gonadotropin-Releasing Hormone Neurons in Mice

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, IA, and a sustained current, IK. Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either IA or IK in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, IA and IK were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased IA in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced IA regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized IA activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.

Activation of Protein Kinase C Delta following Cerebral Ischemia Leads to Release of Cytochrome C from the Mitochondria via Bad Pathway

Background The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release. Methods/Findings We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC. Conclusions We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.

A case report - Volatile metabolomic signature of malignant melanoma using matching skin as a control

Melanoma is the most serious form of skin cancer. The quest for melanoma diagnostic biomarkers is paramount since early detection of melanoma and surgical excision represent the only effective treatment of this capricious disease. Our recent study tested the hypothesis that melanoma forms a unique volatile signature that is different than control, healthy tissue. Here, we are reporting a case study, the analysis of the volatile metabolic signature of a malignant melanoma using matched, non-neoplastic skin tissue from the same patient as a control. This is a significant improvement in the methodology, since it is well known that diet, skin type, genetic background, age, sex and environment all contribute to individual variation in the skin volatile signature. In the present study, we have identified 32 volatile compounds; 9 volatile compounds were increased in melanoma when compared to normal skin and 23 volatile compounds were detected only in melanoma and not in normal skin. Out of these 32 compounds, 10 have been reported previously by our group, thus confirming our results and adding additional confidence in our untargeted metabolomics approach for detection of melanoma biomarkers.

Challenges and opportunities for characterizing cognitive aging across species.

The gradual decline of cognitive ability with age, even in the absence of overt brain disease, is a growing problem. Although cognitive aging is a common and feared accompaniment of the aging process, its underlying mechanisms are not well understood and there are no highly effective means to prevent it. Additional research on cognitive aging is sorely needed, and methods that enable ready translation between human subjects and animal models stand to provide the most benefit. Here and in the six companion pieces in this special issue, we discuss a variety of challenges and opportunities for studying cognitive aging across species. We identify tests of associative memory, recognition memory, spatial and contextual memory, and working memory and executive function as cognitive domains that are age-sensitive and amenable to testing with parallel means in both humans and animal models. We summarize some of the important challenges in using animal models to test cognition. We describe unique opportunities to study cognitive aging in human subjects, such as those provided by recent large-scale initiatives to characterize cognition in large groups of subjects across the lifespan. Finally, we highlight some of the challenges of studying cognitive aging in human subjects.

Chronic nicotine exposure inhibits estrogen-mediated synaptic functions in hippocampus of female rats

Nicotine, the addictive agent in cigarettes, reduces circulating estradiol-17β (E₂) and inhibits E₂-mediated intracellular signaling in hippocampus of female rats. In hippocampus, E₂-signaling regulates synaptic plasticity by phosphorylation of the N-methyl-D-aspartic acid receptor subunit NR2B and cyclic-AMP response element binding protein (pCREB). Therefore, we hypothesized that chronic nicotine exposure induces synaptic dysfunction in hippocampus of female rats. Female rats were exposed to nicotine or saline for 16 days followed by electrophysiological analysis of hippocampus. Briefly, population measurements of excitatory post-synaptic field potentials (fEPSPs) were recorded from stratum radiatum of the CA1 hippocampal slice subfield. A strict software-controlled protocol was used which recorded 30 min of baseline data (stimulation rate of 1/min), a paired-pulse stimulation sequence followed by tetanic stimulation, and 1h of post-tetanus recording. EPSP amplitude and the initial EPSP slope were measured off-line. We then investigated by Western blot analysis the effects of nicotine on hippocampal estrogen receptor-beta (ER-β), NR2B and pCREB. The results demonstrated significantly decreased post-tetanic potentiation and paired-pulse facilitation at the 40, and 80 ms interval in nicotine-exposed rats compared to the saline group. Western blot analysis revealed that nicotine decreased protein levels of ER-β, NR2B, and pCREB. We also confirmed the role of E₂ in regulating NR2B and pCREB phosphorylation by performing Western blots in hippocapmal tissue obtained from E₂-treated ovariectomized rats. In conclusion, chronic nicotine exposure attenuates short-term synaptic plasticity, and the observed synaptic defects might be a consequence of loss of estradiol-17β-signaling. However, determining the exact molecular mechanisms of chronic nicotine exposure on synaptic plasticity specific to the female brain require further investigation.