Volodymyr Gerzanich

Assembly of Human Neuronal Nicotinic Receptor α5 Subunits with α3, β2, and β4 Subunits

Nicotinic acetylcholine receptors formed from combinations of α3, β2, β4, and α5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human α3, β2, β4, and α5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of α3β2, α3β4, α3β2α5, and α3β4α5 subunits but not for other combinations of one, two, or three of these subunits. α5 subunits assembled inside the oocyte with β2 but not with α3 subunits or other α5 subunits. α5 subunits coassembled very efficiently with α3β2 or α3β4 combinations. The presence of α5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also α3 and β2 or α3 and β4 subunits. The presence of α5 subunits increased the rate of desensitization of both receptors containing also α3 and β2 or α3 and β4 subunits. In the case of receptors containing α3 and β4 subunits, the addition of α5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing α3 and β2 subunits, the addition of α5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 μM and the EC50 for nicotine from 6.8 to 1.9 μM, while increasing the efficacy of nicotine from 50% on α3β2 receptors to 100% on α3β2α5 receptors. Both α3β2 and α3β2α5 receptors expressed in oocytes sedimented at the same 11 S value as native α3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma α3, β2, and α5 subunits were co-assembled, and 56% of the receptor subtypes containing α3 subunits also contained β2 subunits. The β2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from α3 and β2 or α3, β2, and α5 subunits rather than the low affinity exhibited by receptors formed from α3 and β4 or α3, β4, and α5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing α3 subunits in SH-SY5Y cells. The affinities of α3β2 receptors expressed in oocytes were similar to the affinities of native α3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Newly expressed SUR1-regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke.

Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NCCa-ATP channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocytes and capillaries. Upregulation of SUR1 was linked to activation of the transcription factor Sp1 and was associated with expression of functional NCCa-ATP but not KATP channels. Block of SUR1 with low-dose glibenclamide reduced cerebral edema, infarct volume and mortality by 50%, with the reduction in infarct volume being associated with cortical sparing. Our findings indicate that the NCCa-ATP channel is crucially involved in development of cerebral edema, and that targeting SUR1 may provide a new therapeutic approach to stroke.

Glibenclamide reduces inflammation, vasogenic edema, and caspase-3 activation after subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) causes secondary brain injury due to vasospasm and inflammation. Here, we studied a rat model of mild-to-moderate SAH intended to minimize ischemia/hypoxia to examine the role of sulfonylurea receptor 1 (SUR1) in the inflammatory response induced by SAH. mRNA for Abcc8, which encodes SUR1, and SUR1 protein were abundantly upregulated in cortex adjacent to SAH, where tumor-necrosis factor-α (TNFα) and nuclear factor (NF)κB signaling were prominent. In vitro experiments confirmed that Abcc8 transcription is stimulated by TNFα. To investigate the functional consequences of SUR1 expression after SAH, we studied the effect of the potent, selective SUR1 inhibitor, glibenclamide. We examined barrier permeability (immunoglobulin G, IgG extravasation), and its correlate, the localization of the tight junction protein, zona occludens 1 (ZO-1). SAH caused a large increase in barrier permeability and disrupted the normal junctional localization of ZO-1, with glibenclamide significantly reducing both effects. In addition, SAH caused large increases in markers of inflammation, including TNFα and NFκB, and markers of cell injury or cell death, including IgG endocytosis and caspase-3 activation, with glibenclamide significantly reducing these effects. We conclude that block of SUR1 by glibenclamide may ameliorate several pathologic effects associated with inflammation that lead to cortical dysfunction after SAH.

Endothelial sulfonylurea receptor 1–regulated NCCa-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury

Acute spinal cord injury (SCI) causes progressive hemorrhagic necrosis (PHN), a poorly understood pathological process characterized by hemorrhage and necrosis that leads to devastating loss of spinal cord tissue, cystic cavitation of the cord, and debilitating neurological dysfunction. Using a rodent model of severe cervical SCI, we tested the hypothesis that sulfonylurea receptor 1-regulated (SUR1-regulated) Ca(2+)-activated, [ATP](i)-sensitive nonspecific cation (NC(Ca-ATP)) channels are involved in PHN. In control rats, SCI caused a progressively expansive lesion with fragmentation of capillaries, hemorrhage that doubled in volume over 12 hours, tissue necrosis, and severe neurological dysfunction. SUR1 expression was upregulated in capillaries and neurons surrounding necrotic lesions. Patch clamp of cultured endothelial cells exposed to hypoxia showed that upregulation of SUR1 was associated with expression of functional SUR1-regulated NC(Ca-ATP) channels. Following SCI, block of SUR1 by glibenclamide or repaglinide or suppression of Abcc8, which encodes for SUR1 by phosphorothioated antisense oligodeoxynucleotide essentially eliminated capillary fragmentation and progressive accumulation of blood, was associated with significant sparing of white matter tracts and a 3-fold reduction in lesion volume, and resulted in marked neurobehavioral functional improvement compared with controls. We conclude that SUR1-regulated NC(Ca-ATP) channels in capillary endothelium are critical to development of PHN and constitute a major target for therapy in SCI.

Hemorrhagic Progression of a Contusion after Traumatic Brain Injury: A Review

The magnitude of damage to cerebral tissues following head trauma is determined by the primary injury, caused by the kinetic energy delivered at the time of impact, plus numerous secondary injury responses that almost inevitably worsen the primary injury. When head trauma results in a cerebral contusion, the hemorrhagic lesion often progresses during the first several hours after impact, either expanding or developing new, non-contiguous hemorrhagic lesions, a phenomenon termed hemorrhagic progression of a contusion (HPC). Because a hemorrhagic contusion marks tissues with essentially total unrecoverable loss of function, and because blood is one of the most toxic substances to which the brain can be exposed, HPC is one of the most severe types of secondary injury encountered following traumatic brain injury (TBI). Historically, HPC has been attributed to continued bleeding of microvessels fractured at the time of primary injury. This concept has given rise to the notion that continued bleeding might be due to overt or latent coagulopathy, prompting attempts to normalize coagulation with agents such as recombinant factor VIIa. Recently, a novel mechanism was postulated to account for HPC that involves delayed, progressive microvascular failure initiated by the impact. Here we review the topic of HPC, we examine data relevant to the concept of a coagulopathy, and we detail emerging data elucidating the mechanism of progressive microvascular failure that predisposes to HPC after head trauma.

De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury

The role of transient receptor potential M4 (Trpm4), an unusual member of the Trp family of ion channels, is poorly understood. Using rodent models of spinal cord injury, we studied involvement of Trpm4 in the progressive expansion of secondary hemorrhage associated with capillary fragmentation, the most destructive mechanism of secondary injury in the central nervous system. Trpm4 mRNA and protein were abundantly upregulated in capillaries preceding their fragmentation and formation of petechial hemorrhages. Trpm4 expression in vitro rendered COS-7 cells highly susceptible to oncotic swelling and oncotic death following ATP depletion. After spinal cord injury, in vivo gene suppression in rats treated with Trpm4 antisense or in Trpm4−/− mice preserved capillary structural integrity, eliminated secondary hemorrhage, yielded a threefold to fivefold reduction in lesion volume and produced a substantial improvement in neurological function. To our knowledge, this is the first example of a Trp channel that must undergo de novo expression for manifestation of central nervous system pathology.

Protective Effect of Delayed Treatment With Low-Dose Glibenclamide in Three Models of Ischemic Stroke

Background and Purpose— Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied. Methods— We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride. Results— Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling. Conclusions— Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.

Sulfonylurea receptor 1 in central nervous system injury: a focused review

The sulfonylurea receptor 1 (Sur1)-regulated NCCa-ATP channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of ‘accidental necrotic cell death’ in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NCCa-ATP channel in acute ischemic, traumatic, and inflammatory injury to the CNS.

Molecular pathophysiology of cerebral edema

Advancements in molecular biology have led to a greater understanding of the individual proteins responsible for generating cerebral edema. In large part, the study of cerebral edema is the study of maladaptive ion transport. Following acute CNS injury, cells of the neurovascular unit, particularly brain endothelial cells and astrocytes, undergo a program of pre- and post-transcriptional changes in the activity of ion channels and transporters. These changes can result in maladaptive ion transport and the generation of abnormal osmotic forces that, ultimately, manifest as cerebral edema. This review discusses past models and current knowledge regarding the molecular and cellular pathophysiology of cerebral edema.

Glibenclamide reduces hippocampal injury and preserves rapid spatial learning in a model of traumatic brain injury.

Cognitive disturbances after traumatic brain injury (TBI) are frequent, even when neuroimaging shows no overt hemorrhagic or other abnormality. Sulfonylurea receptor 1 (SUR1) plays a key role in various forms of CNS injury, but its role in hippocampal dysfunction after mild to moderate TBI is unknown. To assess the hypothesis that postinjury SUR1 upregulation in the hippocampus is associated with a later disturbance in learning, we studied a rat model of cortical impact TBI calibrated to avoid primary and secondary hemorrhage in the underlying hippocampus. The transcription factor, specificity protein 1, which regulates expression of SUR1 and caspase-3, was activated in the hippocampus 15 minutes after injury. Upregulation of SUR1 protein and of Abcc8 (which encodes SUR1) messenger RNA was evident by 6 hours. To assess the role of SUR1, injured rats were administered vehicle or a low dose of the specific sulfonylurea inhibitor glibenclamide for 1 week. At 2 weeks, the increase in activated caspase-3 in the hilus of glibenclamide-treated rats was half of that in vehicle-treated rats. Testing for rapid learning in a Morris water maze at 4 weeks showed significantly better performance in glibenclamide-treated rats; performance inversely correlated with Fluoro-Jade staining for degenerated neurons in the hilus. We conclude that glibenclamide may have long-term protective effects on the hippocampus after mild-to-moderate TBI.