Peter S. Vosler

Calpain-Mediated Signaling Mechanisms in Neuronal Injury and Neurodegeneration

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. Pathologic activation of calpain results in the cleavage of a number of neuronal substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms. In this review, we examine the mechanistic underpinnings of calcium dysregulation resulting in calpain activation in the acute neurodegenerative diseases such as cerebral ischemia and in the chronic neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis, prion-related encephalopathy, and amylotrophic lateral sclerosis. The premise of this paper is that analysis of the signaling and transcriptional consequences of calpain-mediated cleavage of its various substrates for any neurodegenerative disease can be extrapolated to all of the neurodegenerative diseases vulnerable to calcium dysregulation.

Protective effects and mechanisms of sirtuins in the nervous system

Silent information regulator two proteins (sirtuins or SIRTs) are a group of histone deacetylases whose activities are dependent on and regulated by nicotinamide adenine dinucleotide (NAD(+)). They suppress genome-wide transcription, yet upregulate a select set of proteins related to energy metabolism and pro-survival mechanisms, and therefore play a key role in the longevity effects elicited by calorie restriction. Recently, a neuroprotective effect of sirtuins has been reported for both acute and chronic neurological diseases. The focus of this review is to summarize the latest progress regarding the protective effects of sirtuins, with a focus on SIRT1. We first introduce the distribution of sirtuins in the brain and how their expression and activity are regulated. We then highlight their protective effects against common neurological disorders, such as cerebral ischemia, axonal injury, Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and multiple sclerosis. Finally, we analyze the mechanisms underlying sirtuin-mediated neuroprotection, centering on their non-histone substrates such as DNA repair enzymes, protein kinases, transcription factors, and coactivators. Collectively, the information compiled here will serve as a comprehensive reference for the actions of sirtuins in the nervous system to date, and will hopefully help to design further experimental research and expand sirtuins as therapeutic targets in the future.

Hsp27 Protects against Ischemic Brain Injury via Attenuation of a Novel Stress-Response Cascade Upstream of Mitochondrial Cell Death Signaling

Heat shock protein 27 (Hsp27), a recently discovered member of the heat shock protein family, is markedly induced in the brain after cerebral ischemia and other injury states. In non-neuronal systems, Hsp27 has potent cell death-suppressing functions. However, the mechanism of Hsp27-mediated neuroprotection has not yet been elucidated. Using transgenic and viral overexpression of Hsp27, we investigated the molecular mechanism by which Hsp27 exerts its neuroprotective effect. Overexpression of Hsp27 conferred long-lasting tissue preservation and neurobehavioral recovery, as measured by infarct volume, sensorimotor function, and cognitive tasks up to 3 weeks following focal cerebral ischemia. Examination of signaling pathways critical to neuronal death demonstrated that Hsp27 overexpression led to the suppression of the MKK4/JNK kinase cascade. While Hsp27 overexpression did not suppress activation of an upstream regulatory kinase of the MKK/JNK cascade, ASK1, Hsp27 effectively inhibited ASK1 activity via a physical association through its N-terminal domain and the kinase domain of ASK1. The N-terminal region of Hsp27 was required for neuroprotective function against in vitro ischemia. Moreover, knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade effectively inhibited cell death following neuronal ischemia. This underscores the importance of this kinase cascade in the progression of ischemic neuronal death. Inhibition of PI3K had no effect on Hsp27-mediated neuroprotection, suggesting that Hsp27 does not promote cell survival via activation of PI3K/Akt. Based on these findings, we conclude that overexpression of Hsp27 confers long-lasting neuroprotection against ischemic brain injury via a previously unexplored association and inhibition of ASK1 kinase signaling.

Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways.

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca(2+) dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca(2+) homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.

Cellular NAD Replenishment Confers Marked Neuroprotection Against Ischemic Cell Death Role of Enhanced DNA Repair

Background and Purpose— NAD+ is an essential cofactor for cellular energy production and participates in various signaling pathways that have an impact on cell survival. After cerebral ischemia, oxidative DNA lesions accumulate in neurons because of increased attacks by ROS and diminished DNA repair activity, leading to PARP-1 activation, NAD+ depletion, and cell death. The objective of this study was to determine the neuroprotective effects of NAD+ repletion against ischemic injury and the underlying mechanism. Methods— In vitro ischemic injury was induced in rat primary neuronal cultures by oxygen-glucose deprivation (OGD) for 1 to 2 hours. NAD+ was replenished by adding NAD+ directly to the culture medium before or after OGD. Cell viability, oxidative DNA damage, and DNA base-excision repair (BER) activity were measured quantitatively up to 72 hours after OGD with or without NAD+ repletion. Knockdown of BER enzymes was achieved in cultures using AAV-mediated transfection of shRNA. Results— Direct NAD+ repletion in neurons either before or after OGD markedly reduced cell death and OGD-induced accumulation of DNA damage (AP sites, single and double strand breaks) in a concentration- and time-dependent manner. NAD+ repletion restored nDNA repair activity by inhibiting serine-specific phosphorylation of the essential BER enzymes AP endonuclease and DNA polymerase-β. Knocking down AP endonuclease expression significantly reduced the prosurvival effect of NAD+ repletion. Conclusion— Cellular NAD+ replenishment is a novel and potent approach to reduce ischemic injury in neuronal cultures. Restoration of DNA repair activity via the BER pathway is a key signaling event mediating the neuroprotective effect of NAD+ replenishment.

Amphetamine sensitization impairs cognition and reduces dopamine turnover in primate prefrontal cortex

BACKGROUND Amphetamine (AMPH) sensitization in monkeys produces long-lasting behavioral changes that model positive (hallucinatory-like behaviors) and negative (psychomotor depression) symptoms of schizophrenia. The extent to which this model produces the core deficit in schizophrenia--working memory impairment--is unknown. METHODS Two groups of rhesus monkeys were sensitized to AMPH over 6 weeks. In one group, acquisition of cognitive tasks (delayed response, visual discrimination, delayed nonmatch-to-sample) was examined beginning 6+ months postsensitization. The second group was pretrained to stability on delayed response before sensitization. Regional postmortem concentrations of dopamine and its metabolites were examined in tissue from age-matched AMPH-naive and AMPH-sensitized monkeys using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). RESULTS The AMPH-sensitized monkeys were profoundly impaired in their ability to acquire cognitive tasks compared with AMPH-naïve monkeys. Pretrained monkeys showed impaired delayed response performance for several months following sensitization. Analysis by HPLC revealed that AMPH sensitization significantly reduced dopamine turnover in prefrontal cortex and striatum. CONCLUSIONS Impairments in the acquisition and performance of spatial delayed response in association with reduced dopamine turnover in prefrontal cortex following AMPH sensitization provide further support for the relevance of this model to both the etiology and the treatment of cognitive dysfunction in schizophrenia.

Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats.

Traumatic brain injury (TBI) is one of the leading causes of neurological disability in young adults. Edaravone, a novel synthetic small-molecule free-radical scavenger, has been shown to have a neuroprotective effect in both animal models of cerebral ischemia and stroke patients; however, the underlying mechanism is poorly understood. In this report, we investigated the potential mechanisms of edaravone treatment in a rat model of TBI. TBI was induced in the right cerebral cortex of male adult rats using Feeneys weight-drop method. Edaravone (0.75, 1.5, or 3 mg/kg) or vehicle (normal saline) was intravenously administered at 2 and 12 h after TBI. Edaravone treatment significantly decreased hippocampal CA3 neuron loss, reduced oxidative stress, and decreased neuronal programmed cell death compared to vehicle treatment. The protective effects of edaravone treatment were also related to the pathology of TBI on non-neuronal cells, as edaravone decreased astrocyte and glial activation. Lastly, edaravone treatment significantly reduced the presence of inflammatory cytokines, cerebral edema, blood-brain barrier (BBB) permeability, and, importantly, neurological deficits following TBI. Our results suggest that edaravone exerts a neuroprotective effect in the rat model of TBI. The likely mechanism is via inhibiting oxidative stress, leading to a decreased inflammatory response and glial activation, and thereby reducing neuronal death and improving neurological function.

Mitochondrial Targets for Stroke Focusing Basic Science Research Toward Development of Clinically Translatable Therapeutics

Background and Purpose— Stroke is a major cause of death and disability, and it is imperative to develop therapeutics to mitigate stroke-related injury. Despite many promising prospects, attempts at translating neuroprotective agents that show success in animal models of stroke have resulted in very limited clinical success. Summary of Review— This review discusses reasons for the lack of translational success based on the therapeutic targets tested and the pathophysiology of stroke. New recanalization therapies and alternative therapeutic strategies are discussed concerning mitochondria-mediated cell death. Mitochondrial death-regulation pathways are divided into 3 categories: Upstream signaling pathways, agents that target mitochondria directly, and downstream death-execution effectors. The apoptosis signal-related kinase/c-Jun–terminal kinase pathway is used as an example to provide rationale as to why inhibiting signaling pathway upstream of mitochondrial dysfunction is a promising therapeutic approach. Finally, the mechanisms of autophagy and mitochondrial biogenesis are discussed in relation to stroke. Conclusions— Increasing evidence suggests that reperfusion is necessary for improved neurological outcomes after stroke. Development of improved recanalization methods with increased therapeutic windows will aid in improving clinical outcome. Adjunct neuroprotective interventions must also be developed to ensure maximal brain tissue salvage. Targeting prodeath signaling pathways upstream of mitochondrial damage is promising for potential clinically effective treatment. Further understanding of the roles of equilibrium of autophagy and mitochondrial biogenesis in the pathogenesis of stroke could also lead to novel therapeutics.

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

Inducible DNA repair via the base-excision repair pathway is an important prosurvival mechanism activated in response to oxidative DNA damage. Elevated levels of the essential base-excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 correlate closely with neuronal survival against ischemic insults, depending on the CNS region, protective treatments, and degree of insult. However, the precise mechanisms by which this multifunctional protein affords protection and is activated by upstream signaling pathways in postischemic neurons are not well delineated. Here we show that intracerebral administration of pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenously occurring small neuropeptide, induces expression of APE1 in hippocampal neurons. Induction of APE1 expression requires PKA- and p38-dependent phosphorylation of cAMP response-element binding and activating transcription factor 2, which leads to transactivation of the APE1 promoter. We further show that PACAP markedly reduces oxidative DNA stress and hippocampal CA1 neuronal death following transient global ischemia. These effects occurred, at least in part, via enhanced APE1 expression. Furthermore, the DNA repair function of APE1 was required for PACAP-mediated neuroprotection. Thus, induction of DNA repair enzymes may be a unique strategy for neuroprotection against hippocampal injury.

Transgenic Overexpression of Peroxiredoxin-2 Attenuates Ischemic Neuronal Injury Via Suppression of a Redox-Sensitive Pro-Death Signaling Pathway

AIMS Peroxiredoxins (PRXs) are a newly characterized family of peroxide scavenging enzymes that not only help maintain cellular redox homeostasis but also may directly engage in a variety of intracellular signaling pathways. PRX2 is a neuronal-specific PRX believed to participate in cerebral antioxidant responses in several neurodegenerative diseases. This study investigates the potential neuroprotective effect and the underlying mechanism of PRX2 in models of ischemic neuronal injury. RESULTS Transgenic mice overexpressing PRX2 showed reduced brain injury and improved neurological recovery up to 3 weeks after transient focal cerebral ischemia compared to wild-type littermates. In primary cultures of cortical neurons, transfection of PRX2 but not the loss-of-catalytic-site PRX2 mutant conferred neuroprotection against cell death induced by oxygen glucose deprivation. PRX2 exhibited potent pro-survival effects in ischemic neurons by maintaining thioredoxin (Trx) in its reduced state, thereby preventing oxidative stress-mediated activation of apoptosis signal-regulating kinase 1 (ASK1) and the downstream MKK/JNK pro-death signaling pathway. PRX2 failed to provide additional neuroprotection against ischemic injury in Trx- or ASK1-knockdown neuron cultures and in mice treated with a JNK inhibitor. INNOVATION This study provides evidence that neuronal overexpression of PRX2 confers prolonged neuroprotection against ischemic/reperfusion brain injury. Moreover, the results suggest a signaling pathway by which PRX2 suppresses ischemia-induced neuronal apoptosis. CONCLUSIONS Enhanced neuronal expression and activity of PRX2 protect against ischemic neuronal injury by directly modulating the redox-sensitive Trx-ASK1 signaling complex.