Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Neuroprotection against staurosporine by metalloporphyrins independent of antioxidant capability

Metalloporphyrin catalytic antioxidants are remarkably useful in protecting cells and tissues in a wide array of disease models, attributed primarily to functioning as superoxide dismutase (SOD) mimetics or by scavenging other reactive oxygen species (ROS). However, we recently showed that neuroprotection against Ca\ub2\u207a-dependent excitotoxic insults did not correlate with antioxidant strength or capability [25], raising the question of whether scavenging of ROS underlies neuroprotection in other types of neuronal injury. The protein kinase inhibitor staurosporine causes neuronal demise primarily by apoptosis. Neuroprotection from staurosporine by a limited number of metalloporphyrin antioxidants has previously been attributed to antioxidant action. In the current study, a wide array of anionic and cationic metalloporphyrins and porphyrins, ranging in antioxidant strength or capability, provided protection against staurosporine in cortical neuron and cerebellar granule neuron (CGN) culture. Neuroprotection did not correlate with antioxidant strength or capability. In CGN but not cortical neuron cultures, NMDA receptor antagonists also prevented neurotoxicity, so metalloporphyrins may also target this secondary mode of death induced by staurosporine. Neuroprotection observed with antioxidant-inactive controls raises the possibility of an additional, or perhaps alternative, mechanism by antioxidant analogs not involving ROS scavenging.Peer reviewed: YesNRC publication: Ye

Requirement for preclinical prioritization of neuroprotective strategies in stroke: Incorporation of preconditioning

International audienceAcute neuroprotection in numerous human clinical trials has been an abject failure. Major systemic-and procedural-based issues have subsequently been identified in both clinical trials and preclinical animal model experimentation. As well, issues related to the neuroprotective moiety itself have contributed to clinical trial failures, including late delivery, mono-targeting, low potency and poor tolerability. Conditioning (pre-or post-) strategies can potentially address these issues and are therefore gaining increasing attention as approaches to protect the brain from cerebral ischemia. In principle, conditioning can address concerns of timing (preconditioning could be pre-emptively applied in high-risk patients, and post-conditioning after patients experience an unannounced brain infarction) and signaling (multi-modal). However, acute neuroprotection and conditioning strategies face a common translational issue: a myriad of possibilities exist, but with no strategy to select optimal candidates. In this review, we argue that what is required is a neuroprotective framework to identify the "best" agent(s), at the earliest investigational stage possible. This may require switching mindsets from identifying how neuroprotection can be achieved to determining how neuroprotection can fail, for the vast majority of candidates. Understanding the basis for failure can in turn guide supplementary treatment, thereby forming an evidence-based rationale for selecting combinations of therapies. An appropriately designed in vitro (neuron culture, brain slices) approach, based on increasing the harshness of the ischemic-like insult, can be useful in identifying the "best" conditioner or acute neuroprotective therapy, as well as how the two modalities can be combined to overcome individual limitations. This would serve as a base from which to launch further investigation into therapies required to protect the neurovascular unit in in vivo animal models of cerebral ischemia. Based on these respective approaches, our laboratories suggest that there is merit in examining synaptic activity-and nutraceutical-based preconditioning / acute neuroprotection

Preconditioning induces tolerance by suppressing glutamate release in neuron culture ischemia models

This study determined how preconditioned neurons responded to oxygen-glucose deprivation (OGD) to result in neuroprotection instead of neurotoxicity. Neurons preconditioned using chronically elevated synaptic activity displayed suppressed elevations in extracellular glutamate ([glutamateex]) and intracellular Ca\ub2+ (Ca\ub2+ in) during OGD. The glutamate uptake inhibitor TBOA induced neurotoxicity, but at a longer OGD duration for preconditioned cultures, suggestive of delayed up-regulation of transporter activity relative to non-preconditioned cultures. This delay was attributed to a critically attenuated release of glutamate, based on tolerance observed against insults mimicking key neurotoxic signaling during OGD (OGD-mimetics). Specifically, in the presence of TBOA, preconditioned neurons displayed potent protection to the OGD-mimetics: ouabain (a Na+/K+ ATPase inhibitor), high 55 mM KCl extracellular buffer (plasma membrane depolarization), veratridine (a Na+ ionophore), and paraquat (intracellular superoxide producer), which correlated with suppressed [glutamateex] elevations in the former two insults. Tolerance by preconditioning was reversed by manipulations that increased [glutamateex], such as by exposure to TBOA or GABAA receptor agonists during OGD, or by exposure to exogenous NMDA or glutamate. Pre-synaptic suppression of neuronal glutamate release by preconditioning, possibly via suppressed exocytic release, represents a key convergence point in neuroprotection during exposure to OGD and OGD-mimetics. \ua9 2012 National Research Council Canada. Journal of Neurochemistry \ua9 2012 International Society for Neurochemistry.Peer reviewed: YesNRC publication: Ye

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levelsNRC publication: Ye