John McLaughlin

Herpes simplex viral vectors expressing Bcl-2 are neuroprotective when delivered after a stroke.

Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system for the purpose of decreasing necrotic neuronal injury. To that end, we have previously shown that a herpes simplex virus (HSV) vector expressing Bcl-2 could protect neurons from ischemia. In that study, vector was delivered before the ischemia. However, for such gene therapy to be of clinical use, vectors must be protective even if delivered after the onset of the insult. In the present study, we show that an HSV vector expressing Bcl-2 protects striatal neurons when delivered after focal ischemia. Rats were exposed to middle cerebral artery occlusion for 1 hour, followed by reperfusion, and damage was assessed 48 hours later. Delivery of the Bcl-2 vector 30 minutes after reperfusion (i.e., 1.5 hours after ischemia onset) prevented any significant loss of virally-targeted neurons in the striatum. In contrast, in rats microinfused with a vector only expressing a reporter gene, a highly significant loss of neurons occurred. By 4 hours into the reperfusion period (5 hours after ischemia onset), delivery of the Bcl-2 vector was no longer protective. These data show the efficacy of postinsult gene therapy strategies for the brain, underline the finite length of this temporal therapeutic window, and support the growing evidence attesting to the neuroprotective potential of Bcl-2.

Calbindin D28K Overexpression Protects Striatal Neurons From Transient Focal Cerebral Ischemia

Background and Purpose— Increased intracellular calcium accumulation is known to potentiate ischemic injury. Whether endogenous calcium-binding proteins can attenuate this injury has not been clearly established, and existing data are conflicting. Calbindin D28K (CaBP) is one such intracellular calcium buffer. We investigated whether CaBP overexpression is neuroprotective against transient focal cerebral ischemia. Methods— Bipromoter, replication-incompetent herpes simplex virus vectors that encoded the genes for cabp and, as a reporter gene, lacZ were used. Sprague-Dawley rats received bilateral striatal injections of viral vector 12 to 15 hours before ischemia onset. With the use of an intraluminal occluding suture, animals were subjected to 1 hour of middle cerebral artery occlusion followed by 47 hours of reperfusion. Brains were harvested and stained with X-gal (to visualize &bgr;-galactosidase, the gene product of lacZ). The number of remaining virally transfected, X-gal-stained neurons in both the ischemic and contralateral striata were counted and expressed as the percentage of surviving neurons in the ischemic striatum relative to the contralateral nonischemic striatum. Results— Striatal neuron survivorship among cabp-injected animals was 53.5±4.1% (n=10) versus 26.8±5.4% among those receiving lacZ (n=9) (mean±SEM;P <0.001). Conclusions— We conclude that viral vector-mediated overexpression of CaBP leads to neuroprotection in this model of central nervous system injury. This is the first demonstration that CaBP overexpression protects neurons in a focal stroke model.

Calbindin D28K gene transfer via herpes simplex virus amplicon vector decreases hippocampal damage in vivo following neurotoxic insults

Abstract : Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) can lead to neuron death. Preventing a rise in [Ca2+]i by removing Ca2+ from the extracellular space or by adding Ca2+ chelators to the cytosol of target cells ameliorates the neurotoxicity associated with [Ca2+]i increases. Another potential route of decreasing the neurotoxic impact of Ca2+ is to overexpress one of the large number of constitutive calcium‐binding proteins. Previous studies in this laboratory demonstrated that overexpression of the gene for the calcium‐binding protein calbindin D28K, via herpes simplex virus (HSV) amplicon vector, increases the survival of hippocampal neurons in vitro following energetic or excitotoxic insults but not following application of sodium cyanide. We now report that in vivo hippocampal infection with the calbindin D28K HSV vector increases neuronal survival in the dentate gyrus after application of the antimetabolite 3‐acetylpyridine and increases transsynaptic neuronal survival in area CA3 following kainic acid neurotoxicity. The protective effects of infection with the calbindin D28K vector in an intact brain may prove to be beneficial during changes in Ca2+ homeostasis caused by neurological trauma associated with aging and certain neurological diseases.

Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system.

Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS. Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous. To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline. Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold. Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h. In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h. In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively. Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter. Such decline impeded further induction of expression by withdrawing tetracycline. This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors. The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.

Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures

Glucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non‐energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.

Gene therapies that enhance hippocampal neuron survival after an excitotoxic insult are not equivalent in their ability to maintain synaptic transmission

Research shows that overexpression of cytoprotective genes can spare neurons from necrotic death, but few studies have addressed the functional status of surviving neurons. Overexpression of a brain glucose transporter, Glut-1, or the anti-apoptotic protein, Bcl-2, in rats decreases the size of hippocampal lesions produced by kainic acid (KA) treatment. In animals in which KA-induced lesions are reduced to similar extents by Glut-1 or Bcl-2 overexpression, spatial learning is spared by Glut-1, but not Bcl-2. We postulated that Glut-1 and Bcl-2 act differently to protect hippocampal function and investigated the effects of vector overexpression on synaptic physiology after KA treatment. Three days after KA and vector delivery to the dentate gyrus, mossy fiber-CA3 (MF-CA3) population excitatory postsynaptic potentials (EPSPs) were recorded in vitro. In addition to producing a lesion in area CA3, KA treatment reduced baseline MF-CA3 synaptic strength, posttetanic potentiation (PTP), and long-term potentiation (LTP). A similar reduction in the KA-induced lesion was produced by overexpression of Glut-1 or Bcl-2. Glut-1, but not Bcl-2, attenuated the impairments in synaptic strength and PTP. Overexpression of Glut-1 or Bcl-2 preserved LTP after KA treatment. Results indicate greater protection of MF-CA3 synaptic transmission with overexpression of Glut-1 compared to Bcl-2 and suggest that not all neuroprotective gene therapy techniques are equivalent in their ability to spare function.

Effect of GP120 on glutathione peroxidase activity in cortical cultures and the interaction with steroid hormones

GP120 (the protein component of the HIV viral coat) is neurotoxic and may contribute to the cell loss associated with AIDS‐related dementia. Previously, it has been shown in rat cortical mixed cultures that gp120 increased the accumulation of hydrogen peroxide and superoxide, two reactive oxygen species (ROS). We now demonstrate that gp120 increased activity of the key antioxidant glutathione peroxidase (GSPx), presumably as a defensive mechanism against the increased ROS load. Both estrogen and glucocorticoids (GCs), the adrenal steroid released during stress, blunted this gp120 effect on GSPx activity. The similar effects of estrogen and of GCs are superficially surprising, given prior demonstrations that GCs exacerbated and estrogens protected against gp120 neurotoxicity. We find that these similar effects of estrogen and GCs on GSPx regulation arose, in fact, from very different routes, which are commensurate with these prior reports. Specifically, estrogen has demonstrated antioxidant properties that may prevent the ROS increase (therefore acting as a neuroprotective agent) and rendered unnecessary the compensatory GSPx increased activity. To verify this we have added H2O2 to estrogen + gp120‐treated cells, and GSPx activity was increased. However, with addition of H2O2 to GCsu2003+u2003gp120‐treated cells there was no increase in activity. GCs appeared to decrease enzyme production and or activity and therefore under insult conditions ROS levels rose in the cell resulting in increased neurotoxicity. Overexpression of GSPx enzyme via herpes vector system reversed the GCs‐induced loss of enzyme and eliminated the GCs exacerbation of gp120 neurotoxicity.

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.

Delivery of herpes simplex virus amplicon-based vectors to the dentate gyrus does not alter hippocampal synaptic transmission in vivo.

Herpes simplex virus type-1 (HSV) amplicon vectors containing neuroprotective genes can alter cell physiology and enhance survival following various insults. However, to date, little is known about effects of viral infection itself (independent of the gene delivered) on neuronal physiology. Electrically-evoked synaptic responses are routinely recorded to measure functional alterations in the nervous system and were used here to assess the potential capability of HSV vectors to disrupt physiology of the hippocampus (a forebrain structure involved in learning that is highly susceptible to necrotic insult, making it a frequent target in gene therapy research). Population excitatory post-synaptic potentials (EPSPs) were recorded in the dentate gyrus (DG) and in area CA3 in vivo 72 h after infusion of an HSV vector expressing a reporter gene (lacZ) or vehicle into the DG. Evoked perforant path (PP-DG) or mossy fiber (MF-CA3) EPSPs slope values measured across input/output (I/O) curves were not altered by infection. Paired-pulse facilitation at either recording site was also unaffected. X-gal-positive granule cells surrounded the recording electrode (PP-DG recording) and stimulating electrode tracts (MF-CA3 recording) in animals that received vector, suggesting that we had measured function, at least in part, in infected neurons. Because of the negative electrophysiological result, we sought to deliver a gene with an HSV amplicon which would affect the measured endpoints, as a positive control. Delivery of calbindin D28kpotentiated PP-DG synaptic strength, indicating that our recording system could detect alterations due to vector expression. Thus, the data indicate that HSV vectors are benign, in regard to effects on synaptic function, and support the use of these vectors as a safe method to deliver selected genes to the central nervous system.

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.