Neuroprotective effects of an adenoviral vector expressing the glucose transporter: a detailed description of the mediating cellular events

Considerable knowledge exists concerning the events mediating neuron death following a necrotic insult; prompted by this, there have now been successful attempts to use gene therapy approaches to protect neurons from such necrotic injury. In many such studies, however, it is not clear what sequence of cellular events connects the overexpression of the transgene with the enhanced survival. We do so, exploring the effects of overexpressing the Glut-1 glucose transporter with an adenoviral vector in hippocampal cultures challenged with the excitotoxin kainic acid (KA). Such overexpression enhanced glucose transport, attenuated the decline in ATP concentrations, decreased the release of excitatory amino acid neurotransmitters, and decreased the total free cytosolic calcium load. Commensurate with these salutary effects, neuronal survival was enhanced with this gene therapy intervention. Thus, the neuroprotective effects of this particular gene therapy occurs within the known framework of the mechanisms of necrotic neuronal injury.     

Excitotoxic neuroprotection and vulnerability with CaMKII inhibition

Aberrant calcium signaling is a common feature of ischemia and multiple neurodegenerative diseases. While activation of calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key event in calcium signaling, its role in excitotoxicity is controversial. Our findings demonstrate neuroprotection in neuronal cultures treated with the small molecule (KN-93) and peptide (tat-AIP and tat-CN21) inhibitors of CaMKII immediately prior to excitotoxic glutamate/glycine insult. Unlike KN-93 which blocks CaMKII activation, but not constitutively active forms of CaMKII, tat-CN21 and tat-AIP significantly reduced excitotoxicity in cultured neurons when applied post-insult. We observed that the neuroprotective effects of tat-CN21 are greatest when applied before the toxic glutamate challenge and diminish with time, with the neuroprotection associated with CaMKII inhibition diminishing back to control 3h post glutamate insult. Mechanistically, tat-CN21 inhibition of CaMKII resulted in an increase in CaMKII activity and the percentage of soluble αCaMKII observed in neuronal lysates 24h following glutamate stimulation. To address the impact of prolonged CaMKII inhibition prior to excitotoxic insult, neuronal cultures were treated with CaMKII inhibitors overnight and then subjected to a sub-maximal excitotoxic insult. In this model, CaMKII inhibition prior to insult exacerbated neuronal death, suggesting that a loss of CaMKII enhances neuronal vulnerability to glutamate. Although changes in αCaMKII or NR2B protein levels are not responsible for this enhanced glutamate vulnerability, this process is blocked by the protein translation inhibitor cycloheximide. In total, the neuroprotection afforded by CaMKII inhibition can be seen as neuroprotective immediately surrounding the excitotoxic insult, whereas sustained CaMKII inhibition produced by excitotoxicity leads to neuronal death by enhancing neuronal vulnerability to glutamate.     

An adenoviral vector expressing the glucose transporter protects cultured striatal neurons from 3-nitropropionic acid

Considerable interest has focused on the possibility of using gene transfer techniques to introduce protective genes into neurons around the time of necrotic insults. We have previously used herpes simplex virus amplicon vectors to overexpress the rat brain glucose transporter, Glut-1 (GT), and have shown it to protect against a variety of necrotic insults both in vitro and in vivo, as well as to buffer neurons from the steps thought to mediate necrotic injury. It is critical to show the specificity of the effects of any such transgene overexpression, in order to show that protection arises from the transgene delivered, rather than from the vector delivery system itself. As such, we tested the protective potential of GT overexpression driven, in this case, by an adenoviral vector, against a novel insult, namely exposure of primary striatal cultures to the metabolic poison, 3-nitropropionic acid (3NP). We observed that GT overexpression buffered neurons from neurotoxicity induced by 3NP.     

Peroxiredoxin 2 overexpression protects cortical neuronal cultures from ischemic and oxidative injury but not glutamate excitotoxicity, whereas Cu/Zn superoxide dismutase 1 overexpression protects only against oxidative injury

We previously reported that peroxiredoxin 2 (PRDX2) and Cu/Zn superoxide dismutase 1 (SOD1) proteins are up-regulated in rat primary neuronal cultures following erythropoietin (EPO) preconditioning. In the present study, we have demonstrated that adenovirally mediated overexpression of PRDX2 in cortical neuronal cultures can protect neurons from in vitro ischemia (oxygen-glucose deprivation) and an oxidative insult (cumene hydroperoxide) but not glutamate excitotoxicity. We have also demonstrated that adenovirally mediated overexpression of SOD1 in cortical neuronal cultures protected neurons only against the oxidative insult. Interestingly, we did not detect up-regulation of PRDX2 or SOD1 protein in the rat hippocampus following exposure to either 3 min or 8 min of global cerebral ischemia. Further characterization of PRDX2's neuroprotective mechanisms may aid in the development of a neuroprotective therapy.     

Relative Resistance of Striatal Neurons Containing Calbindin or Parvalbumin to Quinolinic Acid-Mediated Excitotoxicity Compared to Other Striatal Neuron Types

To evaluate the relative ability of those striatal neuron types containing calbindin or parvalbumin to withstand a Ca²⁺-mediated excitotoxic insult, we injected the NMDA receptor-specific excitotoxin quinolinic acid (QA) into the striatum in mature adult rats and 2 months later examined the relative survival of striatal interneurons rich in parvalbumin and striatal projection neurons rich in calbindin. To provide standardization to the survival of striatal neuron types thought to be poor in Ca²⁺buffering proteins, the survival was compared to that of somatostatin–neuropeptide Y (SS/NPY)-containing interneurons and enkephalinergic projection neurons, which are devoid of or relatively poorer in such proteins. The various neuron types were identified by immunohistochemical labeling for these type-specific markers and their relative survival was compared at each of a series of increasing distances from the injection center. In brief, we found that parvalbuminergic, calbindinergic, and enkephalinergic neurons all showed a generally comparable gradient of neuronal loss, except just outside the lesion center, where calbindin-rich neurons showed significantly enhanced survival. In contrast, striatal SS/NPY interneurons were more vulnerable to QA than any of these three other types. These observed patterns of survival following intrastriatal QA injection suggest that calbindin and parvalbumin content does not by itself determine the vulnerability of striatal neurons to QA-mediated excitotoxicity in mature adult rats. For example, parvalbuminergic striatal interneurons were not impervious to QA, while cholinergic striatal interneurons are highly resistant and SS/NPY+ striatal interneurons are highly vulnerable. Both cholinergic and SS/NPY+ interneurons are devoid of any known calcium buffering protein. Similarly, calbindin does not prevent striatal projection neuron vulnerability to QA excitotoxicity. Nonetheless, our data do suggest that calbindin may offer striatal neurons some protection against moderate excitotoxic insults, and this may explain the reportedly slightly greater vulnerability of striatal neurons that are poor in calbindin to ischemia and Huntington's disease.     

Calbindin‐D28K is increased in the ventral horn of spinal cord by neuroprotective factors for motor neurons

Slow glutamate‐mediated neuronal degeneration is implicated in the pathophysiology of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The calcium‐binding proteins calbindin‐D_28K and parvalbumin have been reported to protect neurons against excitotoxic insults. Expression of calbindin‐D_28K is low in adult human motor neurons, and vulnerable motor neurons additionally may lack parvalbumin. Thus, it has been speculated that the lack of calcium‐binding proteins may, in part, be responsible for early degeneration of the population of motor neurons most vulnerable in ALS. Using a rat organotypic spinal cord slice system, we examined whether the most potent neuroprotective factors for motor neurons can increase the expression of calbindin‐D_28K or parvalbumin proteins in the postnatal spinal cord. After 4 weeks of incubation of spinal cord slices with 1) glial cell line‐derived neurotrophic factor (GDNF), 2) neurturin, 3) insulin‐like growth factor I (IGF‐I), or 4) pigment epithelium‐derived factor (PEDF), the number of calbindin‐D_28K‐immunopositive large neurons (>20 μm) in the ventral horn was higher under the first three conditions, but not after PEDF, compared with untreated controls. Under the same conditions, parvalbumin was not upregulated by any neuroprotective factor. The same calbindin increase was true of IGF‐I and GDNF in a parallel glutamate toxicity model of motor neuron degeneration. Taken together with our previous reports from the same model, which showed that all these neurotrophic factors can potently protect motor neurons from slow glutamate injury, the data here suggest that upregulation of calbindin‐D_28K by some of these factors may be one mechanism by which motor neurons can be protected from glutamate‐induced, calcium‐mediated excitotoxicity. © 2015 Wiley Periodicals, Inc.     

Gene therapy in the nervous system with superoxide dismutase

Neuronal death following necrotic insults involves the generation of reactive oxygen species (ROS). We investigated the effects of antioxidant gene therapy on ROS accumulation after exposure to either sodium cyanide, kainic acid or oxygen glucose deprivation (OGD). Specifically, we generated herpes simplex virus-1 amplicon vector expressing the gene for the antioxidant enzyme CuZnSOD. Overexpression of this gene in primary hippocampal cultures resulted in increased enzymatic activity of the corresponding protein. CuZnSOD significantly protected hippocampal neurons against sodium cyanide insult and the subsequent lipid peroxidation. However, it did not protect against OGD- or kainic-acid-induced toxicity. Moreover, CuZnSOD significantly worsened the toxicity, hydrogen peroxide accumulation and lipid peroxidation induced by kainic acid. As a possible explanation for this surprising worsening, CuZnSOD overexpression increased glutathione peroxidase activity in the presence of sodium cyanide but had no effect on catalase or glutathione peroxidase activity in the presence of kainic acid. Thus, cells were unlikely to be able to detoxify the excess hydrogen peroxide produced as a result of the CuZnSOD overexpression. These studies can be viewed as a cautionary note concerning gene therapy intervention against necrotic insults.     

Effects of ciliary neurotrophic factor on excitotoxicity and calcium-ionophore A23187-induced cell death in cultured embryonic striatal neurons

Ciliary neurotrophic factor (CNTF) has a protective effect on the striatum in animal models of Huntington's disease. However, the mechanism through which it exerts its effect is not clear. In this study, we show that there is a concentration-dependent direct protective effect of CNTF against N-methyl-D-aspartate-mediated excitotoxicity on striatal neurons in vitro. The CNTF has to be added more than half an hour before the insult for the effect to occur and its effect is eliminated by the presence of the protein synthesis inhibitor cycloheximide. This suggests that the protective mechanism of CNTF does not involve acute interference with the glutamate receptors, but probably requires gene/protein expression. We have also shown that the effect of CNTF against glutamate-induced excitotoxicity is dependent on the concentration of glutamate with a protective effect more evident at a low grade excitotoxic insult. Finally, we saw no effect of CNTF on calcium ionophore A23187-induced toxicity in striatal cultures, indicating that the growth factor does not promote survival by enhancing general defenses against raised intracellular levels of calcium.     

Molecular factors underlying selective vulnerability of motor neurons to neurodegeneration in amyotrophic lateral sclerosis

Current research evidence suggests that genetic factors, oxidative stress and glutamatergic toxicity, with damage to critical target proteins and organelles, may be important contributory factors to motor neuron injury in amyotrophic lateral sclerosis (ALS). Various molecular and neurochemical features of human motor neurons may render this cell group differentially vulnerable to such insults. Motor neurons are large cells with long axonal processes which lead to requirements for a high level of mitochondrial activity and a high neurofilament content compared to other neuronal groups. The lack of calcium buffering proteins parvalbum in and calbindin D28k and the low expression of the GluR2 AMPA receptor subunit may render human motor neurons particularly vulnerable to calcium toxicity following glutamate receptor activation. Motor neurons also have a high perisomatic expression of the glutamate transporter protein EAAT2 and a very high expression of the cytosolic free radical scavenging enzyme Cu/Zn superoxide dismutase (SOD1) which may render this cell group vulnerable in the face of genetic or post-translational alterations interfering with the function of these proteins. More detailed characterisation of the molecular features of human motor neurons in the future may allow the strategic development of better neuroprotective therapies for the benefit of patients afflicted by ALS.

     

The neuronal death induced by endotoxic shock but not that induced by excitatory amino acids requires TNF‐α

We studied, using organotypic hippocampal slices in culture, the role of pro‐inflammatory cytokines, oxygen radicals and nitric oxide in neuronal death induced either by endotoxic insult [interferon (IFN) γ, 24 h followed by lipopolysaccharide, 24 h] or by glutamate receptor‐mediated excitotoxic insult. We demonstrated that neuronal death induced by endotoxic insult was absolutely dependent on the synthesis of tumour necrosis factor alpha (TNF‐α). Indeed, TNF‐α antibodies and SB203580, an inhibitor of p38 stress kinase known to block TNF‐α and other cytokine synthesis, completely protected neurons from the endotoxic insult. Inhibiting oxygen radical and nitric oxide productions also reduced the endotoxic shock. We also showed that after priming the cultures with IFN‐γ, TNF‐α was unable to induce neuronal death unless oxygen‐free radicals were exogenously provided. In contrast, although glutamate receptor‐induced excitotoxicity was associated with a low TNF‐α synthesis and a modest activation of p38 stress kinase, neither TNF‐α antibodies nor SB203580 were able to decrease excitotoxic neuronal insult. We did not reduce glutamate receptor‐induced neuronal death with superoxide dismutase plus catalase. In conclusion, although inflammation follows glutamate receptor‐mediated neurotoxicity, the mechanisms by which an endotoxic insult triggers neuronal death are different from those involved in excitotoxicity.     

Neuroprotection with herpes simplex vectors expressing virally derived anti-apoptotic agents

A large body of literature dealing with neurotoxicity has focused on trying to define the exact nature of cell death following a neurological insult. While there is some debate in the field, it has been shown that a number of neurons in a given population can respond to an acute insult stimulus by activating the apoptotic cascade. To what extent, however, these insults result in the classical manifestations of either apoptosis or necrosis, or whether a mixture of the two results, is highly controversial, in part dependent on the particular system utilized. In this paper, we investigate the role of particular apoptotic signals in cultured rat hippocampal neurons, following acute excitotoxicity, metabolic poisoning, and heat stress. In particular, we examine these effects by utilizing a modified herpes simplex viral vector to specifically deliver viral anti-apoptotic genes. We have selected a battery of viral genes (crmA, p35, gamma34.5, KsBcl-2) that have evolved to suppress suicidal host responses to infection. We examine these inhibitors in the face of the above classes of insults and report that each viral agent tested has a unique profile in its ability to protect hippocampal neurons following acute neurological insults. Specifically, the effects of domoic acid excitotoxicity can be alleviated only with crmA, p35 and gamma34.5 whereas all genes tested can protect against heat stress. Conversely, no genes tested can protect against metabolic poisoning by cyanide. Such a study helps us to further understand the nature of apoptotic signals in different insults.     

Ca2+-ATPase isoforms are expressed in neuroprotection in rat, but not human, neurons

Glutamate excitotoxicity has been implicated in neuronal death and damage in many neurodegenerative disorders. The potential neuroprotective role of the plasma membrane calcium ATPase (PMCA) and the NMDA receptor were investigated in rat and human brain neurons after a glutamate insult. Investigation of potential mechanisms of neuronal survival revealed that surviving rat cerebellar granule cells expressed the mRNA of new PMCA isoforms 2b and 2c. There was no observable change in expression of PMCA isoforms or NMDA receptor NR2 subtypes in human cortical neurons. This study shows that subsets of rat and human neurons are resistant to glutamate-induced excitotoxicity and the mechanisms employed to enable survival differ between rat and human neurons.     

Modeling of acquired postischemic epileptogenesis in cultures of neural cells and tissue

We reviewed the literature data on the cellular and molecular mechanisms of postischemic epileptogenesis, which is modeled in a culture of dissociated neurons and organotypic culture of nervous tissue. We summarized the data on the involvement of glutamate excitotoxicity in the development of epileptiform discharges generated by neurons in culture after glutamate application and on the involvement of neuronal calcium overload in the formation of seizure activity. We also presented data on the effects of neuroprotectors in models of postischemic epileptogenesis in vitro.     

Hypoxic/ischaemic cell damage in cultured human NT-2 neurons

Postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line in a novel rapid differentiation procedure. These neurons were used to establish an in vitro assay system that allows the investigation of hypoxic/ischaemic cell damage and the development of neuroprotective strategies. In experiments of simulated ischaemia, the neurons were subjected to anoxia and hypoglycaemia. The viability of NT-2 neuronal cells was significantly reduced by anoxia especially in the presence of glutamate, reflecting the cellular vulnerability to excitotoxic conditions. The addition of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduced glutamate-induced neuronal damage. Calcium imaging showed that NT-2 neurons increased cytosolic calcium levels in response to stimulation with glutamate or NMDA, an effect that was abolished in calcium free medium and at low pH values. The NMDA receptor antagonists MK-801, AP 5 and ketamine reduced the NMDA-induced response, suggesting the presence of functional NMDA receptors in the human neuronal cells. The mitochondrial potential of neurons was estimated using the fluorescent dye rhodamine 123 (R123). The fluorescence imaging experiments indicated an energetic collapse of mitochondrial functions during anoxia, suggesting that the human NT-2 neurons can be used to investigate subcellular processes during the excitotoxic cascade.     

Gene therapy for treatment of cerebral ischemia using defective herpes simplex viral vectors

Significant advances have been made over the past few years concerning the cellular and molecular events underlying neuron death. Recently, it is becoming increasingly clear that some of the genes induced during cerebral ischemia may actually serve to rescue the cell from death. However, the injured cell may not be capable of expressing protein at levels high enough to be protective. One of the most exciting arenas of such interventions is the use of viral vectors to deliver potentially neuroprotective genes at high levels. Neurotrophic herpes simplex viral strains are an obvious choice for gene therapy to the brain, and we have utilized bipromoter vectors that are capable of transferring various genes to neurons. Using this system in experimental models of stroke, cardiac arrest and excitotoxicity, we have found that it is possible to enhance neuron survival against such cerebral insults by over-expressing genes that target various facets of injury. These include energy restoration by the glucose transporter (GLUT-1), buffering calcium excess by calbindin, preventing protein malfolding or aggregation by stress proteins and inhibiting apoptotic death by BCL-2. We show that in some cases, gene therapy is also effective after the onset of injury, and also address whether successful gene therapy necessarily spares function. Although gene therapy is limited to the few hundred cells the vector is capable of transfecting, we consider the possibility of such gene therapy becoming relevant to clinical neurology in the future.     

Baclofen is neuroprotective and prevents loss of calcium/calmodulin-dependent protein kinase II immunoreactivity in the ischemic gerbil hippocampus

Excessive release of glutamate during transient cerebral ischemia initiates a cascade of events that leads to the delayed and selective death of neurons located in the hippocampus. Activity of calcium calmodulin kinase II (CaM kinase), a protein kinase critical to neuronal functioning, disappears following ischemia. The in vivo link between glutamate excitoxicity and alterations in CaM kinase activity has not been extensively studied. Baclofen, a selective gamma-aminobutyric acid (GABA)(B) receptor agonist, has been shown to inhibit glutamate release. The present study evaluated the neuroprotective efficacy of this compound and assessed early changes in hippocampal-dependent behaviors and CaM kinase immunoreactivity following transient cerebral ischemia. Baclofen (50 mg/kg) prevented both the loss of hippocampal CA1 pyramidal cells and the reduction in hippocampal CaM kinase immunoreactivity observed in control animals following ischemic insult. Cerebral ischemia produced a significant increase in working memory errors; however, baclofen failed to attenuate this memory deficit. Results confirm that baclofen is neuroprotective and support a link between glutamate excitotoxicity and reductions in CaM kinase immunoreactivity.     

Phenformin suppresses calcium responses to glutamate and protects hippocampal neurons against excitotoxicity

Phenformin is a biguanide compound that can modulate glucose metabolism and promote weight loss and is therefore used to treat patients with type-2 diabetes. While phenformin may indirectly affect neurons by changing peripheral energy metabolism, the possibility that it directly affects neurons has not been examined. We now report that phenformin suppresses responses of hippocampal neurons to glutamate and decreases their vulnerability to excitotoxicity. Pretreatment of embryonic rat hippocampal cell cultures with phenformin protected neurons against glutamate-induced death, which was correlated with reduced calcium responses to glutamate. Immunoblot analyses showed that levels of the N-methyl-d-aspartate (NMDA) subunits NR1 and NR2A were significantly decreased in neurons exposed to phenformin, whereas levels of the AMPA receptor subunit GluR1 were unchanged. Whole-cell patch clamp analyses revealed that NMDA-induced currents were decreased, and AMPA-induced currents were unchanged in neurons pretreated with phenformin. Our data demonstrate that phenformin can protect neurons against excitotoxicity by differentially modulating levels of NMDA receptor subunits in a manner that decreases glutamate-induced calcium influx. These findings show that phenformin can modulate neuronal responses to glutamate, and suggest possible use of phenformin and related compounds in the prevention and/or treatment of neurodegenerative conditions.     

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca2+]i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca2+]i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.     

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture,and against focal ischemic brain injury in vivo

Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions. J. Neurosci. Res. 53:613–625, 1998. © 1998 Wiley-Liss, Inc.     

Intracellular calcium signals in the surround of rat visual cortex lesions

Focal lesions of the visual cortex induce deafferentiation, excitotoxic cell death as well as functional reorganization in the surrounding tissue. The intracellular second messenger calcium is involved in a wide range of cellular responses including excitotoxicity and functional reorganization following cortical injuries. We investigated the intracellular calcium concentration [Ca2+]i in neurons of the visual cortex using fluorescence imaging of fura-2 signals in a slice preparation obtained from lesioned and sham-operated cortices. We observed an increase in resting and stimulus evoked [Ca2+]i in the surround of the lesion, which were mediated by NMDA and non-NMDA ionotropic glutamate receptors. This increase in [Ca2+]i might be an important factor for lesion-induced functional reorganization in the rat visual cortex.