Michael Tymianski

Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity

Excitotoxicity contributes to neuronal degeneration in many acute CNS diseases, including ischemia, trauma, and epilepsy, and may also play a role in chronic diseases, such as amyotrophic lateral sclerosis (ALS). Key mediators of excitotoxic damage are Ca ions (Ca(2+)), which under physiological conditions govern a multitude of cellular processes, including cell growth, differentiation, and synaptic activity. Consequently, homeostatic mechanisms exist to maintain a low intracellular Ca(2+) ion concentration so that Ca(2+) signals remain spatially and temporally localized. This permits multiple independent Ca-mediated signaling pathways to occur in the same cell. In excitotoxicity, excessive synaptic release of glutamate can lead to the disregulation of Ca(2+) homeostasis. Glutamate activates postsynaptic receptors, including the ionotropic N-methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) proprionate (AMPA), and kainate receptors. Upon their activation, these open their associated ion channel to allow the influx of Ca(2+) and Na(+) ions. Although physiological elevations in intracellular Ca(2+) are salient to normal cell functioning, the excessive influx of Ca(2+) together with any Ca(2+) release from intracellular compartments can overwhelm Ca(2+)-regulatory mechanisms and lead to cell death. Although Ca(2+) disregulation is paramount to neurodegeneration, the exact mechanism by which Ca(2+) ions actually mediate excitotoxicity is less clear. One hypothesis outlined in this review suggests that Ca(2+)-dependent neurotoxicity occurs following the activation of distinct signaling cascades downstream from key points of Ca(2+) entry at synapses, and that triggers of these cascades are physically co-localized with specific glutamate receptors. Thus, we summarize the importance of Ca(2+) regulation in mammalian neurons and the excitotoxicity hypothesis, and focus on the molecular determinants of glutamate receptor-mediated excitotoxic mechanisms.

A Key Role for TRPM7 Channels in Anoxic Neuronal Death

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role.

Glutamate receptors, neurotoxicity and neurodegeneration

Glutamate excitotoxicity is a hypothesis that states excessive glutamate causes neuronal dysfunction and degeneration. As glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), the implications of glutamate excitotoxicity are many and far-reaching. Acute CNS insults such as ischaemia and traumatic brain injury have traditionally been the focus of excitotoxicity research. However, glutamate excitotoxicity has also been linked to chronic neurodegenerative disorders such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease and others. Despite the continued research into the mechanisms of excitotoxicity, there are currently no pharmacological interventions capable of providing significant neuroprotection in the clinical setting of brain ischaemia or injury. This review addresses the current state of excitotoxic research, focusing on the structure and physiology of glutamate receptors; molecular mechanisms underlying excitotoxic cell death pathways and their interactions with each other; the evidence for glutamate excitotoxicity in acute neurologic diseases; laboratory and clinical attempts at modulating excitotoxicity; and emerging targets for excitotoxicity research.

NMDA Receptor Subunits Have Differential Roles in Mediating Excitotoxic Neuronal Death Both In Vitro and In Vivo

Well-documented experimental evidence from both in vitro and in vivo models of stroke strongly supports the critical involvement of NMDA receptor-mediated excitotoxicity in neuronal damage after stroke. Despite this, the results of clinical trials testing NMDA receptor antagonists as neuroprotectants after stroke and brain trauma have been discouraging. Here, we report that in mature cortical cultures, activation of either synaptic or extrasynaptic NR2B-containing NMDA receptors results in excitotoxicity, increasing neuronal apoptosis. In contrast, activation of either synaptic or extrasynaptic NR2A-containing NMDA receptors promotes neuronal survival and exerts a neuroprotective action against both NMDA receptor-mediated and non-NMDA receptor-mediated neuronal damage. A similar opposing action of NR2B and NR2A in mediating cell death and cell survival was also observed in an in vivo rat model of focal ischemic stroke. Moreover, we found that blocking NR2B-mediated cell death was effective in reducing infarct volume only when the receptor antagonist was given before the onset of stroke and not 4.5 h after stroke. In great contrast, activation of NR2A-mediated cell survival signaling with administration of either glycine alone or in the presence of NR2B antagonist significantly attenuated ischemic brain damage even when delivered 4.5 h after stroke onset. Together, the present work provides a molecular basis for the dual roles of NMDA receptors in promoting neuronal survival and mediating neuronal damage and suggests that selective enhancement of NR2A-containing NMDA receptor activation with glycine may constitute a promising therapy for stroke.

Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury.

Stroke and neurotrauma mediate neuronal death through a series of events that involve multiple interdependent molecular pathways. It has been suggested that these pathways are triggered following elevations in extracellular excitatory amino acids, primarily glutamate [1]. This report outlines mechanisms involving glutamate-mediated excitotoxicity with specific focus on (i) the role of Ca2+ in neurotoxicity, (ii) The concept of ‘source specificity’ of neurotoxicity, (iii) the role of the ionotropic N-methyl-D-aspartate (NMDA)-subtype glutamate receptor and its associated submembrane molecules that may give rise to signaling specificity in excitotoxicity and (iv) the role of glutamate-mediated free-radical generation and associated cell death pathways. We also highlight a novel, peptide-based approach for uncoupling NMDA receptors from excitotoxicity in the rat central nervous system subjected to focal ischemia, thereby reducing stroke infarct volume and improving neurological functioning.

Molecular Mechanisms of Glutamate Receptor-Mediated Excitotoxic Neuronal Cell Death

Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca2+) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca2+-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.

Calcium, ischemia and excitotoxicity.

The initial reports regarding a cytotoxic role of calcium ions were published over 30 years ago. In neurons, calcium ions can gain entry into the cell through several mechanisms. These include the over-activation of glutamate receptors (NMDA, AMPA, KA) or of a range of channels and transporters (TRPM2, TRPM7, NCX, ASICs, CaV1.2, and hemichannels). Potentially toxic cytoplasmic calcium concentrations can also occur due to release from internal stores, either through physical damage to mitochondria and the endoplasmic reticulum, or a malfunction of receptors and channels present in their membranes. Such increases of cytoplasmic calcium concentrations can trigger a range of downstream neurotoxic cascades, including the uncoupling mitochondrial electron transfer from ATP synthesis, and the activation and overstimulation of enzymes such as calpains and other proteases, protein kinases, nitric oxide synthase (NOS), calcineurin and endonucleases. Despite the toxic role of calcium, drugs designed to block its entry into neurons have all failed to have any beneficial effects in clinical trials. We suggest that blocking certain receptors and ion channels is unlikely to be a useful therapeutic strategy due to potential deleterious side effects. However, identifying those that are most responsible for cell death and their downstream signalling pathways may lead to improved strategies for treating ischemic and excitotoxic disorders.

MOLECULAR MECHANISMS OF CALCIUM DEPENDENT EXCITOTOXICITY

Abstract. Excitotoxicity is thought to be a major mechanism contributing to neurodegeneration during central nervous system ischemia, trauma, and other neurological disorders. Briefly, synaptic overactivity leads to the excessive release of glutamate, the major excitatory neurotransmitter in the mammalian central nervous system. Glutamate activates a number of postsynaptic cell membrane receptors, which upon activation open their associated ion channel pore to produce ion influx or efflux. This leads to a disturbance of the intracellular ionic environment, the best characterized feature of which is the influx of sodium, chloride, and Ca2+. An excess of Ca2+ ions then activates intracellular Ca2+-dependent signaling cascades that eventually lead to neuronal cell death. Despite intensive research in the field of Ca2+-dependent neurotoxicity the precise molecular mechanisms leading to cell death remain poorly understood. In particular, the question of the precise relationship between Ca2+ loading and neurotoxicity has been controversial. Many glutamate receptors are clustered and localized at the postsynaptic density. Recently, increasing knowledge of the molecular composition of the postsynaptic density has allowed us to extend our understanding of the molecular mechanisms of Ca2+-dependent excitotoxicity and to propose that distinct, membrane receptor-specific, neurotoxic signaling pathways transduce Ca2+-dependent excitotoxicity. These findings may have significant implications in the search for precisely targeted therapeutic drugs for a range of neurological disorders.

Normal and Abnormal Calcium Homeostasis in Neurons: A Basis for the Pathophysiology of Traumatic and Ischemic Central Nervous System Injury

Clinical recovery after central nervous system (CNS) trauma or ischemia may be limited by a neural injury process that is triggered and perpetuated at the cellular level, rather than by a lesion amenable to surgical repair. It is widely thought that one such process, a fundamental pathological mechanism initiated by CNS injury, is a disruption of cellular Ca2+ homeostasis. Because of the critical role of Ca2+ ions in regulating innumerable cellular functions, this major homeostatic disturbance is thought to trigger neuronal and axonal degeneration and produce clinical disability. We review those aspects of normal and pathological Ca2+ homeostasis in neurons that relate to neurodegeneration and to the application of neuroprotective strategies for the treatment of CNS injury. In particular, we examine the contribution of Ca(2+)-permeable ionic channels, Ca2+ pumps, intracellular Ca2+ stores, intracellular Ca2+ buffering systems, and the roles of secondary, Ca(2+)-dependent processes in neurodegeneration. A number of hypotheses linking Ca2+ ions and Ca2+ permeable channels to neurotoxicity are discussed with an emphasis on strategies for lessening Ca(2+)-related damage. A number of these strategies may have a future role in the treatment of traumatic and ischemic CNS injury.

The Natural History and Predictive Features of Hemorrhage From Brain Arteriovenous Malformations

Background and Purpose— Patients harboring brain arteriovenous malformations (bAVMs) are at a lifelong risk for hemorrhagic strokes, but the natural history is poorly understood. We examined the impact of demographic and angiographic features on the likelihood of future hemorrhage. Methods— A prospectively accrued database of bAVM patients maintained at the Toronto Western Hospital was analyzed; 678 consecutive, prospectively enrolled bAVM patients were followed for 1931.7 patient-years. The rate of hemorrhage over long-term follow-up was recorded. The impact of baseline clinical and radiographic features and partial treatment on time to hemorrhage were analyzed using survival analysis. Neurological outcome after hemorrhage was assessed using the Glasgow Outcome Score. Results— Hemorrhage rates were 4.61% per year for the entire cohort (n=678), 7.48% per year for bAVMs with initial hemorrhagic presentation (n=258), 4.16% per year for initial seizure presentation (n=260), 3.99% per year for patients not harboring aneurysms (n=556), 6.93% per year for patients with associated aneurysms (n=122), and 5.42% per year for bAVMs with deep venous drainage (n=365). Hemorrhagic presentation was a significant independent predictor of future hemorrhage (HR, 2.15; P<0.01), whereas associated aneurysms (HR, 1.59; P=0.07) and deep venous drainage (HR, 1.59; P=0.07) showed a trend toward significance. Hemorrhage risk was unchanged in patients who underwent partial arteriovenous malformation embolization (n=211; HR, 0.875; P=0.32). Conclusion— Brain arteriovenous malformations presenting with hemorrhage, with deep venous drainage, or associated aneurysms have ≈2-fold greater likelihood of a future hemorrhage. Partial treatment by embolization does not alter these risks. This natural history should be taken into account in the treatment strategy.