Metabolomics Based Identification of SIRT5 and Protein Kinase C Epsilon Regulated Pathways in Brain.

The role of Sirtuins in brain function is emerging, yet little is known about SIRT5 in this domain. Our previous work demonstrates that protein kinase C epsilon (PKCε)-induced protection from focal ischemia is lost in SIRT5-/- mice. Thus, metabolic regulation by SIRT5 contributes significantly to ischemic tolerance. The aim of this study was to identify the SIRT5-regulated metabolic pathways in the brain and determine which of those pathways are linked to PKCε. Our results show SIRT5 is primarily expressed in neurons and endothelial cells in the brain, with mitochondrial and extra-mitochondrial localization. Pathway and enrichment analysis of non-targeted primary metabolite profiles from Sirt5-/- cortex revealed alterations in several pathways including purine metabolism (urea, adenosine, adenine, xanthine), nitrogen metabolism (glutamic acid, glycine), and malate-aspartate shuttle (malic acid, glutamic acid). Additionally, perturbations in ß-oxidation and carnitine transferase (pentadecanoic acid, heptadecanoic acid) and glutamate transport and glutamine synthetase (urea, xylitol, adenine, adenosine, glycine, glutamic acid) were predicted. Metabolite changes in SIRT5-/- coincided with alterations in expression of amino acid (SLC7A5, SLC7A7) and glutamate (EAAT2) transport proteins as well as key enzymes in purine (PRPS1, PPAT), fatty acid (ACADS, HADHB), glutamine-glutamate (GAD1, GLUD1), and malate-aspartate shuttle (MDH1) metabolic pathways. Moreover, PKCε activation induced alternations in purine metabolites (urea, glutamine) that overlapped with putative SIRT5 pathways in WT but not in SIRT5-/- mice. Finally, we found that purine metabolism is a common metabolic pathway regulated by SIRT5, PKCε and ischemic preconditioning. These results implicate Sirt5 in the regulation of pathways central to brain metabolism, with links to ischemic tolerance.

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

BACKGROUND AND PURPOSE: Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. METHODS: We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. RESULTS: Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1, accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. CONCLUSIONS: Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders.

Physical Exercise Improves Cognitive Outcomes in 2 Models of Transient Cerebral Ischemia.

BACKGROUND AND PURPOSE: Preclinical studies suggest that exercise can enhance cognition after cerebral ischemia but often use long training regiments and test cognition during or acutely after training. The cognitive changes may result from enhanced physical fitness and may only provide acute benefit. We sought to determine whether a short period of exercise after cerebral ischemia could improve cognitive outcomes when measured days after completion of exercise training in 2 cerebral ischemia models. METHODS: Focal or global cerebral ischemia was induced in Sprague-Dawley rats. Rats recovered (3-4 days) then were subject to no exercise (0 m/min), mild (6 m/min), moderate (10 m/min), or heavy (15-18 m/min) treadmill exercise (5-6 days). Cognition was tested 8 to 10 days after the last exercise session with hippocampal-dependent contextual fear conditioning. RESULTS: A short training period of moderate exercise enhanced cognitive function for a week after exercise completion in both models of cerebral ischemia. CONCLUSIONS: Utilization of this exercise paradigm can further the elucidation of exercise-mediated factors involved in cognitive recovery independent of changes in physical fitness.

Protein kinase C epsilon delays latency until anoxic depolarization through arc expression and GluR2 internalization.

Global cerebral ischemia is a debilitating injury that damages the CA1 region of the hippocampus, an area important for learning and memory. Protein kinase C epsilon (PKCɛ) activation is a critical component of many neuroprotective treatments. The ability of PKCɛ activation to regulate AMPA receptors (AMPARs) remains unexplored despite the role of AMPARs in excitotoxicity after brain ischemia. We determined that PKCɛ activation increased expression of a protein linked to learning and memory, activity-regulated cytoskeleton-associated protein (arc). Also, arc is necessary for neuroprotection and confers protection through decreasing AMPAR currents via GluR2 internalization. In vivo, activation of PKCɛ increased arc expression through a BDNF/TrkB pathway, and decreased GluR2 mRNA levels. In hippocampal cultured slices, PKCɛ activation decreased AMPAR current amplitudes in an arc- and GluR2-dependent manner. Additionally, PKCɛ activation triggered an arc- and GluR2 internalization-dependent delay in latency until anoxic depolarization. Inhibiting arc also blocked PKCɛ-mediated neuroprotection against lethal oxygen and glucose deprivation. These data characterize a novel PKCɛ-dependent mechanism that for the first time defines a role for arc and AMPAR internalization in conferring neuroprotection.

Cognitive Deficits after Cerebral Ischemia and Underlying Dysfunctional Plasticity: Potential Targets for Recovery of Cognition.

Cerebral ischemia affects millions of people worldwide and survivors suffer from long-term functional and cognitive deficits. While stroke and cardiac arrest are typically considered when discussing ischemic brain injuries, there is much evidence that smaller ischemic insults underlie neurodegenerative diseases, including Alzheimer's disease. The "regenerative" capacity of the brain relies on several aspects of plasticity that are crucial for normal functioning; less affected brain areas may take over function previously performed by irreversibly damaged tissue. To harness the endogenous plasticity mechanisms of the brain to provide recovery of cognitive function, we must first understand how these mechanisms are altered after damage, such as cerebral ischemia. In this review, we discuss the long-term cognitive changes that result after cerebral ischemia and how ischemia alters several plasticity processes. We conclude with a discussion of how current and prospective therapies may restore brain plasticity and allow for recovery of cognitive function, which may be applicable to several disorders that have a disruption of cognitive processing, including traumatic brain injury and Alzheimer's disease.