Potassium channel gene therapy can prevent ... - Wiley Online Library

Journal of Neurochemistry, 2003

doi:10.1046/j.1471-4159.2003.01880.x

Potassium channel gene therapy can prevent neuron death resulting from necrotic and apoptotic insults Angela L. Lee, Theodore C. Dumas, Phiroz E. Tarapore, Brian R. Webster, Dora Y. Ho, Daniela Kaufer and Robert M. Sapolsky Department of Biological Sciences, Stanford University, Stanford, California, USA

Abstract Necrotic insults such as seizure are excitotoxic. Logically, membrane hyperpolarization by increasing outwardly conducting potassium channel currents should attenuate hyperexcitation and enhance neuron survival. Therefore, we overexpressed a small-conductance calcium-activated (SK2) or voltage-gated (Kv1.1) channel via viral vectors in cultured hippocampal neurons. We found that SK2 or Kv1.1 protected not only against kainate or glutamate excitotoxicity but also increased survival after sodium cyanide or staurosporine. In vivo overexpression of either channel in dentate gyrus reduced kainate-induced CA3 lesions. In hippocampal slices, the kainate-induced increase in granule cell excitability was reduced by overexpression of either channel, suggesting that these channels exert their protective effects during hyperex-

citation. It is also important to understand any functional disturbances created by transgene overexpression alone. In the absence of insult, overexpression of Kv1.1, but not SK2, reduced baseline excitability in dentate gyrus granule cells. Furthermore, while no behavioral disturbances during spatial acquisition in the Morris water maze were observed with overexpression of either channel, animals overexpressing SK2, but not Kv1.1, exhibited a memory deficit post-training. This difference raises the possibility that the means by which these channel subtypes protect may differ. With further development, potassium channel vectors may be an effective pre-emptive strategy against necrotic insults. Keywords: central nervous system, herpes simplex virus, neurotoxicity, rodent, seizure. J. Neurochem. (2003) 10.1046/j.1471-4159.2003.01880.x

Necrotic insults such as seizure can result in neuron death in vulnerable hippocampal regions. Hyperexcitability of neurons which have suffered insult is central to their eventual death since it leads to massive elevation of the highly excitatory neurotransmitter, glutamate. This in turn leads to abberant activation of excitatory synapses and excessive mobilization of free cytosolic calcium (Ca2+) in postsynaptic neurons. This results in a number of calcium-dependent degenerative events, eventually culminating in necrosis (Choi 1994) or, in a subset of neurons, apoptosis (Bredesen 1995). Outwardly rectifying potassium (K+) channels act to regulate neuronal activity. In hippocampal neurons, voltage-gated K+ channel subtypes (Kv) have been shown to be involved in the repolarization of action phase potentials (Meir et al. 1999) and, in turn, alter the probability of glutamate transmitter release (Perreault and Avoli 1991; Dorandeu et al. 1997). Calcium-activated channels (SK) are more efficacious in modulating the number of energyexpensive action potentials (Attwell and Laughlin 2001) discharged during depolarization by mediating various afterhyperpolarizations (AHPs) (Sah 1996). This is reflected

in spike frequency adaptation, which is the lengthening of the time between action potentials in a burst, brought about by lengthening the opening time of SK channels (Madison and Nicoll 1984). Enhancing such regulation during and excitotoxic insult to diminish hyperexcitability should therefore be protective. Openers of endogenous K+ channels protect against ischemia-induced depolarization (Ben-Ari 1990; Gribkoff et al. 2001) and reduce neuron death following cerebral artery occlusion (Heurteaux et al. 1993; Gribkoff et al.

Resubmitted manuscript received April 7, 2003; accepted April 7, 2003. Address correspondence and reprint requests to Angela L. Lee, Department of Biological Sciences, Stanford University, Stanford, CA 94305–5020, USA. E-mail: [email protected] Abbreviations used: ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarization; EPSP, excitatory postsynaptic potential; hCMV, human cytomegalovirus; HSV, herpes simplex virus; KA, kainic acid; Kv, voltage-gated K+ channel subtype; PBS, phosphate-buffered saline; PF, paraformaldehyde; PP, perforant path; PS, population spike; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SK, calciumactivated channel.

2003 International Society for Neurochemistry, J. Neurochem. (2003) 10.1046/j.1471-4159.2003.01880.x

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2001). Similarly, increasing the number of inwardly rectifying ATP-gated K+ channels (KATP) by adenovirus gene transfer reduces neuronal excitability (Ehrengruber et al. 1997). We tested the protective effects of overexpressing the a subunit of either the mammalian Ca2+-activated SK2 channel (Kohler et al. 1996) or Kv1.1 channel (Christie et al. 1989). A neuron’s rapid Ca2+ influx in response to excess glutamate generated during an insult is a defining feature of glutamate neurotoxicity. Therefore, using a Ca2+-activated K+ channel would exploit one of the earliest events leading to cell death as the trigger for K+ channel function. This subtype of K+ channel is responsible for the slow components of AHP that underlie spike frequency adaptation following action potentials (Madison and Nicoll 1984). Kv1.1 channels are believed to modulate the action potential (Papazian et al. 1987), and their loss can cause epilepsy (Smart et al. 1998). We present data suggesting that overexpression of either SK2 or Kv1.1 channels can protect neurons in vitro and in vivo from necrotic insults, and that, in the absence of any insult, K+ channel overexpression can alter normal physiology and behavior.

Materials and methods Virus preparation Rat Kv1.1 (Christie et al. 1989) or SK2 (Kohler et al. 1996) cDNA, encoding the a subunit of either channel, was subcloned with the human cytomegalovirus (hCMV) ie1 polyA into the unique HindIII site of pa22bgal as a 1916 (Kv1.1) or 2109 (SK2) bp fragment. Each was under the control of the herpes simplex virus (HSV) a4 promoter, and each construct also contained b-galactosidase (bgal) as a reporter gene under the control of the a22 promoter, generating either pa4RBKa22bgal or pa4RSKa22bgal (Fig. 1). These amplicons were packaged into modified replication-deficient HSV vectors that were purified as previously described (Ho 1994). Negative control plasmid was pa4bgal, which expresses the reporter gene only. After packaging these amplicons into modified HSV vectors and purifying the vectors, typical yields were 1–17 · 106 vectors/mL and 2–30 · 106 helper virus particles/mL. With this bipromoter system there is greater than 98% coexpression between the reporter gene and the experimental transgene (Fink et al. 1997). K+ channel overexpression Kinetic RT-PCR. Day 18 fetal mixed hippocampal cultures were established as previously described from rats (Brooke et al. 1997). Primary hippocampal cultures (approximately 20–30% neurons, 70– 80% glia) were infected with 30 000 amplicons/1.8 · 106 cells/well (MOI 0.017) on day 10 after plating into a six-well plate. This amount of vector typically infects at a ratio of 4 : 1 neurons/glia (unpublished observations). Sixteen to 22 hours later (enough time for maximal gene expression), cells were washed once with phosphate-buffered saline (PBS) and total RNA was isolated using an RNeasy mini-prep kit (Qiagen, Valencia, CA, USA) and then

Fig. 1 Kv1.1 or SK2-expressing construct. Plasmids incorporated into HSV amplicons contain the following: a, HSV packaging signal; SV40 polyA, poly(A) signal from Simian virus 40; bgal, b-galactosidase cDNA; a22, HSV alpha22 immediate-early promoter; OriS, HSV origin of replication; a4, HSV alpha4 immediate-early promoter; Kv1.1 or SK2 cDNA; CMV polyA, poly(A) signal from cytomegalovirus ie1 gene.

treating with DNAseI (Invitrogen, Carlsbad, CA, USA). Two hundred nanograms of total RNA were used for PCR per primer pair. Primers used were Kv1.1561(+) 5¢-TCATCCTCATCTCCATAGTCATC-3¢, kv1.11167 (–) 5¢ATGTCACCGTATCCCACAG-3¢, SK2565 (+) 5¢-TACCACGCCAGGGAAATAC-3¢, SK2980 (–) 5¢GCGGCAATTATCCATAACGAG-3¢. PCR conditions were: 30 min at 48C, 2 min at 94C, followed by 36 cycles of 15 s at 94C, 30 s at 57C, 1 min at 72C. Samples were drawn from cycles 21 and on, every third cycle. Ten per cent of the PCR product was run on a 1.5% agarose gel and visualized with ethidium bromide. Densitometry was performed using Photoshop 5.5 (Adobe, San Jose, CA, USA). Western blot. Primary hippocampal cultures were infected at an MOI 0.017 on day 10 after plating into a six-well plate. Sixteen to 22 hours later cells were washed once with PBS and lysed in 4· sample buffer [250 mM Tris-HCl pH 6.8, 8% sodium dodecyl sulfate (SDS), 0.02% bromphenol blue, 0.4 M DTT, 40% glycerol] and boiled for 5 min DNA in samples was sheared by passing the 1 lysate through a 20-gauge needle. Lysates from equal numbers of cells were loaded into lanes of a 7.5% SDS gel with a stack. After electrophoresis, proteins were transferred onto Hybond-P polyvinylidene difluoride (PVDF) transfer membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and blocked in 5% milk in PBS. Blots were washed in PBS/0.2% Tween-20 (Sigma, St. Louis, MO, USA) and incubated with a 1 : 200 dilution of rabbit anti-rat Kv1.1 mAb (Sigma) in 5% milk/0.2% Tween-20. (It was not possible to obtain antibody that demonstrated the presence of SK2 protein, data not shown). Blots were then washed and incubated with 1: 2000 dilution of goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Blots were washed again and incubated with avidin DH/ biotinylated horseradish peroxidase H complex (Vectastain Elite ABC Kit, Vector Laboratories). After final washes, bands were visualized using chemiluminescence (Renaissance Enhanced Luminol Reagent, NEN, Boston, MA, USA). In vitro neurotoxicity assay. Ten days after plating, primary mixed hippocampal cultures were infected at an MOI of 0.017. A maximum


K+ channel gene therapy reduces neurotoxicity

of 45% of neurons can be infected with this amplicon system (Roy et al. 2001). Sixteen to 22 hours later glutamate, staurosporine, or sodium cyanide (all Sigma) or kainic acid (KA, Diagnostic Chemicals Ltd, Charlottetown PEI, Canada) was added to the culture medium. For glutamate experiments, medium was changed after 2 h insult exposure. All insult doses were chosen to bracket an LD50 dose. 24 h after the final manipulation, cultures were methanol-fixed and assayed for neuronal survival by quantifying MAP-2+ cells via an ELISA-based assay (Brooke et al. 1999). In vivo neuroprotection. Adult male (150–250 g) Sprague–Dawley rats were anesthetized (10 : 2 : 1 ketamine/acepromazine/xylazine; 100 : 10 : 100 mg/mL stocks, 1 mL cocktail/kg). An injection volume of 1.9 lL, comprising 1.5 lL vector and 0.4 lL KA (0.1 mg/mL KA stock) was delivered to each hemisphere at a rate of 0.5 lL/min. bgal/KA was injected into the dentate gyrus (DG)/CA3 region of the right or left hippocampus [A/P 3.85, M/L 3.35 from bregma, D/V 2.55 from dura (Paxinos 1998)] and 1.5 lL of Kv1.1/ KA or SK2/KA into the contralateral region. In experimental animals, 0.04 lg of KA was injected in addition to either control or experimental vector. In vivo, reporter expression becomes evident 2–4 h postinfection, reaches a maximal plateau at 12–72 h, and declines to undetectable levels by about 5 days (unpublished observation). CA3 lesions are apparent 72 h following KA. Seventy-two hours after surgery, animals were killed and perfused with heparinized saline followed by 4% paraformaldehyde (PF) in PBS. The brains were dissected and cryoprotected in 4% PF/ 20% sucrose/PBS and then cut in a cryostat to 30 lM coronal sections. Every third section was stained with cresyl violet and assayed for cell loss to the CA3 region under 10 · magnification using a 10 · 10 ocular grid and counting the total number of pixels for area CA3 as well as the number of pixels with damaged or missing cells. Data were reported as the percentage of the CA3 that was lesioned, from at least 10 sections through the dorsal hippocampus. Analyses were performed blind with respect to the infection state. Veterinary care was provided by the AALAC-approved Department of Comparative Medicine at Stanford University. These procedures comply with the recommendations of the Panel on Euthanasia of the American Medical Association and have been approved by Stanford University. In addition, the vivarium is accredited by the Administrative Panel on Laboratory Animal Care. Hippocampal slice electrophysiology Vector (1.5 lL/hemisphere, 0.5 lL/min) was injected bilaterally into the DG of adult male Sprague Dawley rats (4.5 mm A/P, 3.0 mm M/L from bregma, 3.2 mm from dura) under anesthesia (see in vivo neuroprotection). K+ channel vector was delivered to one hemisphere and bgal to the other. In some animals, PBS was delivered bilaterally. Subsequent analyses were performed blind with respect to the infection state. Three days following vector delivery, animals were killed and the brain removed and placed into oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF in mM: NaCl 124, KCl 2, MgSO4 1.7, CaCl2 2.3, KH2PO4 1.25, NaHCO3 26, and dextrose 10, pH 7.4). The hippocampus was dissected free and sliced parallel to the alvear fibers (450 lm). Slices were immediately transferred to an interface recording chamber (1.5 lL/min, room temperature)

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to incubate for at least 2 h prior to recording. Slices used in KA experiments were maintained in oxygenated ACSF at room temperature and transferred individually to the recording chamber. Electrophysiological measurements were made in the superior blade of the DG. To evoke perforant path (PP)-DG excitatory postsynaptic potentials (EPSPs) and population spikes (PSs), a Teflon-insulated stimulating electrode (concentric bipolar, Pt-Ir, 115 lm pole diameter) was placed in contact with the slice surface stratum moleculare to activate medial PP axons. For EPSP recording, a recording electrode (1.5 mW glass pipette filled with ACSF) was placed in the inner third of the molecular layer (determined by the lack of paired-pulse facilitation). To observe the PS, the electrode was moved to the stratum granulosum. In this design, the stimulation intensity was set to evoke a baseline PS of either 0.5 or 1 mV in amplitude. The EPSP slope was calculated as the maximum slope across a 1-ms epoch near the response onset. PS amplitude was determined as the voltage difference between cursors set to define the initial EPSP peak and the minimum negative deflection. In contrast to our recordings in areas CA3 or CA1, medial PP fiber potentials were small and difficult to isolate and were not analyzed. For some slices, a stable baseline was recorded for at least 10 min and then KA (5 lM) was applied to the perfusate for 10 min. Histology. After in vitro recording, slices were transferred to 3% PF for 10–20 min followed by a PBS wash (5–10 min). Slices were stained in X-Gal (Molecular Probes, Eugene, OR, USA) overnight and stored in PF until being embedded in gelatin (300 bloom, 14%, in H2O) and postfixed in PF for at least 48 h. 40 mm sections were cut by vibratome and mounted on slides. Xgal-positive (blue) cells were counted under low-power light microscopy (100 ·). Slices void of Xgal-positive neurons in the DG (i.e. outside the area of infection around the needle track) and the corresponding electrophysiology data were discarded. Behavioral testing Cannulae implantation and injection procedures. Animals were anesthetized (see in vivo neuroprotection) and implanted with bilateral indwelling guide cannulae intended to target the DG (4.0– 4.2 A/P, 3.0 M/L from bregma, 3.0–3.2 D/V from dura). Animals were allowed to recover for 3–4 days, and were then injected bilaterally with Kv1.1, SK2, bgal, or PBS. Vector (1.5 lL) was delivered at a rate of 0.5 lL/min via an injection cannula inserted through the guide cannula. Animals were handled 1 day before and during vector delivery. Behavioral procedures were initiated 24 h after vector delivery. Spatial memory in Morris water maze. This task requires animals to use external sensory cues to find a submerged escape and is dependent on an intact hippocampus (Morris 1984). Training consisted of six blocks (three trials/block) with a 20–30 min interblock interval, all performed on the same day. During training, the hidden platform was stationary while the start location varied pseudo-randomly. 20 min following training, a probe trial (labeled immediate) was performed where the platform was removed from the pool and the animal was allowed to free swim for 1 min Subsequently, three more trials were performed with the platform



replaced. Twenty-four hours after the first probe trial, a second probe trial was performed (labeled 24 h). Animal movement was tracked with a CCD camera (VIM camera 2400, Hamamatsu, Japan) positioned directly above the pool and behavior was analyzed offline (MetaMorph software, Universal Imaging, Downingtown, PA, USA). Mean escape latency and path length to find the platform for each block was calculated for each animal and averaged by vector group to construct learning curves. For the probe trials, dwell time in the goal quadrant was divided by dwell time in the opposite quadrant for each animal and averaged by vector group to produce mean goal/opposite ratios (G : O). Statistics. Statistical analyses for in vitro and in vivo neuroprotection were by two-way ANOVA, as indicated, followed by Tukey post hoc tests. To assess group differences in mean field EPSP slope across the I/O curves two-way ANOVA were performed. One-way ANOVA were used to compare the EPSP slope necessary to evoke a 0.5-mV PS and the change in the EPSP and PS magnitude during KA administration. During behavioral testing, one-way ANOVA were used to analyze for differences between groups at each block and for differences in G : O ratios. SigmaStat 6.0 (Jandel Scientific Software, San Rafael, CA, USA) was used for all statistical analyses.

Results

Comparisons of kinetic RT-PCR product level from K+ channel- and control-infected primary hippocampal cultures (Fig. 2) were used to examine K+ channel mRNA overexpression. Kv1.1 or SK2 mRNA was detected by densitometric analysis (Fig. 2a,b). Actin controls (Fig. 2c) showed no difference in mRNA levels from an endogenous gene in cells infected with any of the three vectors. Western blot analysis demonstrated overexpression of Kv1.1 protein in culture (Fig. 2d). As previously stated, there is greater than 98% coexpression between the reporter gene and the experimental gene in this vector system (Fink et al. 1997). In vivo, bgal enzyme activity as assayed by Xgal staining in brain slices indicated that vectors were expressed mostly in the superior blade of the dentate gyrus (Fig. 2e,f) after infusion of either Kv1.1 or SK2 amplicon. In primary cultures, Kv1.1 or SK2 overexpression 16–22 h pre-insult protected against the excitotoxins glutamate and KA (Fig. 3a,b) as compared with cultures infected with reporter gene alone. For glutamate, Kv1.1 protected over a greater range of doses than SK2. Against KA, both protected at the same doses, while SK2 protected against a greater range of doses against the metabolic inhibitor sodium cyanide (Fig. 3c). It should be noted that vectors did not protect against the highest doses of KA or cyanide, despite those being no more toxic than the next highest doses [KA (F2,197 ¼ 6.663, p < 0.798), cyanide (F2,294 ¼ 12.454, p < 0.93)]; the reason for this is unknown. Both vectors were mildly, although significantly protective against only one dose of staurosporine (Fig. 3d). Moreover,

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 2 Overexpression of Kv1.1 or SK2 K+ channels in vitro and in vivo. (a–c) mRNA as assayed by kinetic PCR from primary cells infected with amplicon using primers specific for (a) SK2; (b) Kv1.1; or (c) actin. PCR products are from cells infected with (a, top panel) SK2, or (a, bottom) bgal; (b, top) Kv1.1 or (b, bottom) bgal; (a) or (c, top) Kv1.1, or (a, bottom) bgal. Aliquots of the PCR were taken after 21, 24, 27, 30, and 33 cycles (SK2 primers, a); 24, 27, 30, 33, and 36 cycles (Kv1.1 primers, b); or 24, 27, 30, and 33 cycles (actin primers, c). Densitometry levels are indicated below each band. (d) Protein as assayed by western blot of primary cultures infected with Kv1.1expressing construct. +, infected with Kv1.1-expressing amplicon; –, infected with bgal-expressing amplicon. (e) In vivo reporter expression: Xgal staining of an SK2-infected brain slice after electrophysiological testing. (f) Xgal staining of a Kv1.1-infected brain slice after electrophysiological testing.

KA-induced lesions in vivo were reduced 77% by SK2, and 37% by Kv1.1 (Fig. 4a,b). We also observed that KAinduced seizures were qualitatively less severe in rats which had received Kv1.1, and to a lesser degree, SK2, compared to rats which received bgal vector (data in preparation). Thus, K+ channel overexpression appears to be neuroprotective both in vitro and in vivo over an array of insult models. In order to explore how K+ channel overexpression might exert its protective effects, we examined the effect of K+ channel overexpression on the excitability increase produced by KA (Collingridge et al. 1983). KA increases EPSP/PS coupling in that for an evoked EPSP of a constant magnitude, the associated PS increases with exposure to KA. In slices that overexpressed bgal only, KA increased the PS roughly



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Fig. 3 Overexpression of Kv1.1 or SK2 K+ channels protects against insults in vitro. Survival is expressed as the percentage of cells surviving after infection with vector and insult/% of cells surviving after vector addition alone. Data are mean ± SEM (n ¼ 18–80). Survival in bgal- and Kv1.1-treated cultures differed significantly by two-way ANOVA [(a) glutamate (F3,328 ¼ 0.909, p < 0.001); (b) kainic acid (F2,131 ¼ 2.213, p < 0.001); (c) sodium cyanide (F2,294 ¼ 5.634, p < 0.05); (d) staurosporine (F1,105 ¼ 0.712, p < 0.02)], as did survival in bgal- and SK2-treated cultures [(a) glutamate (F3,367 ¼ 3.947, p < 0.001); (b) kainic acid (F2,131 ¼ 6.773, p < 0.001); (c) sodium cyanide (F2,294 ¼ 2.056, p < 0.001); (d) staurosporine (F1,107 ¼ 0.000057, p < 0.05)]. Survival of Kv1.1- and SK2-treated cultures did not differ, however (non-significant by two-way ANOVA). *,***Indicate significantly different at p < 0.05, 0.01, respectively, when compared to bgal at the same insult dose by post hoc t-tests.

3.6-fold (Fig. 4c). In slices overexpressing either Kv1.1 or SK2, the KA-induced increase in the PS was decreased to nearly 2.2-fold. Thus, the protection against KA excitotoxicity provided by Kv1.1 or SK2 K+ overexpression in vitro

and in vivo may be due, in part, to a reduction in the initial increase in excitability. To address the effects of K+ channel overexpression on normative function, we examined changes in synaptic transmission and membrane excitability in DG granule cells of hippocampal slices. When recording was performed in the molecular layer 3 days following vector delivery, the EPSP slope increased with increasing stimulation intensity in a similar manner after delivery of PBS, bgal, or Kv1.1. However, the EPSP slope increased more across the I/O curve for slices overexpressing SK2 than for any other group (Fig. 5a). When examining synaptically driven PSs recorded in the granular layer of the DG, we observed that the EPSP necessary to drive a 0.5-mV PS was greater for slices overexpressing Kv1.1 relative to SK2, bgal or PBS (Fig. 5b). These data suggest a reduction in postsynaptic excitability with enhanced Kv1.1 channel function and an increase in medial PP synaptic strength with enhanced SK2 channel function. The findings also suggest that overexpression of different K+ channel subtypes might provide neuroprotection in different ways. Having shown that K+ channel overexpression alters normal neuron function on the population level, we sought to test whether these effects were sufficient to alter behavior. We used the Morris water maze (Morris 1984) as a hippocampus-dependent learning task. Animals that learn the platform location show shorter escape latencies as they are trained. Figure 6(a) shows that pooled controls and animals overexpressing either K+ channel exhibited reduced average escape latencies across training blocks with no statistical difference between groups. To assure that animals learned spatial location of the platform, an immediate probe was performed (Table 1). On average, all groups exhibited goal/opposite ratios greater than 1, indicating preference for the goal quadrant and acquisition of the spatial location of the escape platform. To test memory for platform location, a 24-h probe was performed (Table 1). Pooled controls and animals overexpressing Kv1.1, but not SK2, showed goal/



Fig. 4 Overexpression of Kv1.1 or SK2 K+ channels protects against insults in vivo. Data are mean lesion size ± SEM after simultaneous infusion of 0.04 lg KA and either Kv1.1 [(a) Kv1.1 (F1,15 ¼ 2.552, p < 0.05)] or SK2 [(b) SK2 (F1,17 ¼ 22.964, p < 0.001)]. Data were compared using two-way ANOVA. (c) Potassium channel overexpression reduces the excitation produced by KA. Bar graph shows the percent increase relative to baseline in the PS after delivery of KA to hippocampal slices. bgal, n ¼ 16 slices; Kv1.1 n ¼ 14; SK2 n ¼ 11. *,***Indicate p < 0.05, 0.01, respectively, in post hoc t-tests.

opposite ratios greater than 1 indicating continued preference for the goal quadrant. No goal preference in the SK2 animals suggests that overexpression of the calcium-activated K+ channels disrupts consolidation of spatial information.

Fig. 5 Potassium channel overexpression alters normative physiology in DG granule cells in the hippocampal slice. (a) shows the mean EPSP slope (and SEM) as a function of stimulation intensity for PBS (n ¼ 9 slices), bgal (n ¼ 22), Kv1.1 (n ¼ 16) and SK2 animals (n ¼ 13). A two-way ANOVA for EPSP slope revealed effects of vector (F3,334 ¼ 7.5, p < 0.0001) and stimulation level (F5,334 ¼ 151.7, p < 0.0001) as well as an interaction (F15,334 ¼ 2.4, p < 0.005). (b) shows the mean EPSP slope associated with a 0.5-mV PS. PBS n ¼ 25 slices; bgal n ¼ 25; Kv1.1 n ¼ 21; SK2 n ¼ 11. Inset shows typical mediant perforant path field EPSPs and PSs for each group (average of five individual sweeps). Scale bars are 0.5 mV (vertical) and 10 ms (horizontal). *Indicates p < 0.05 for post hoc t-tests between Kv1.1 and each other group.

Discussion

The hippocampus is particularly vulnerable to necrotic insults such as seizure. Enhancing K+ channel function can protect neurons against necrotic insults (Ben-Ari 1990; Heurteaux et al. 1993; Zini et al. 1993; Gribkoff et al. 2001), presumably by reducing insult-induced hyperexcitability. Concordant with this, we found that Kv1.1 or SK2 gene therapy protected neurons against necrotic insults. In vitro insults included the excitotoxins glutamate and KA, the metabolic poison sodium cyanide, and the apoptosisinducer staurosporine; this represents a broad range of protection compared with most other transgenes tested against necrotic neurological insults (reviewed in Sapolsky

and Steinberg 1999; Sapolsky 2003). When introduced prior to the excitotoxins glutamate and KA, both the Kv1.1 and SK2 channels were protective in vitro. In vivo overexpression of Kv1.1 or SK2 in DG granule cells reduced the CA3 lesion produced by KA providing further evidence of trans-synaptic protection (Lawrence et al. 1995; Phillips et al. 1999; Dumas et al. 2000). KA delivery to the DG depolarizes granule cells and increases the release of glutamate from mossy fiber terminals (Nadler and Cuthbertson 1980) possibly producing damage to CA3 pyramidal cells directly. However, mossy fiber synaptic activation elicits pyramidal cell discharge (Henze et al. 2002)



Fig. 6 In vivo overexpression of Kv1.1 or SK2 does not affect a hippocampal-dependent learning task. Panel shows the escape latency for each training block (three trials per block). Data are mean ± SEM. (‘Control’ is the data from animals injected with either PBS or bgal n ¼ 14; Kv1.1 n ¼ 25; SK2 n ¼ 12). Data from the first six blocks were analyzed by two-way repeated-measures ANOVA, revealing a block (F5,175 ¼ 13.68, p < 0.0001) but no group effect.

Table 1 In vivo overexpression of SK2 alters a hippocampaldependent memory task. Probe trials were run 20 min following the 6th block of training (immediate) and 24 h after the initial probe trial. Data are presented as the ratio of the time spent in the goal quadrant to the time spent in the opposite quadrant (goal/opposite). One-group t-test indicated G : O ratios that were greater than 1 for all groups in the immediate probe and for the pooled control and Kv1.1 groups in the 24 h probe. G : O scores could not be calculated for four animals 4 (one pooled control, 2 Kv1.1 and 1 SK2) Goal/Opposite ± SEM

Control Kv1.1 SK2

Immediate

24 h

3.90* ± 0.60 (n ¼ 14) 4.72* ± 0.91 (n ¼ 11) 4.79* ± 1.34 (n ¼ 12)

2.55* ± 0.66 (n ¼ 14) 2.88* ± 0.61 (n ¼ 11) 1.47 ± 0.23 (n ¼ 10)

*Indicates p < 0.02 for the immediate probe and p < 0.05 for the 24 h probe.

and increased activity at CA3-CA3 collateral synapses which contain NMDA receptors [in contrast, mossy fiber synapses contain very few NMDA receptors (Monaghan et al. 1983)]. It is believed that KA delivery to the DG enhances NMDA receptor activity at CA3-CA3 synapses, thereby toxically mobilizing cytosolic calcium. Consonant with this, KA neurotoxicity in area CA3 is reduced by NMDA antagonists (Lee et al. 2002). As we have shown, overexpression of Kv1.1 or SK2 blunted the KA-induced increases in DG excitability. Therefore, these K+ channels plausibly suppress the chain of events leading to CA3 pyramidal cell hyperactivity and death.

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Vector was introduced prior to insult in vitro, such that overexpression was maximal at the time of insult. In vivo, KA and vector were administered simultaneously. KA causes 12–24 h of status epilepticus seizures within minutes. Transgene expression is first evident 2–4 h postinfection and is maximal at 12–20 h. As such, while K+ channel overexpression did not alter seizure quality 4–6 h post KA/ vector delivery, it did accelerate seizure attenuation 10–24 h post delivery (data in preparation). This suggests that reduction of seizure duration, rather than of magnitude, mediates the neuroprotective effects of overexpression. Overexpression of SK2 or Kv1.1 protected against sodium cyanide. Cyanide-induced energy deprivation can increase extracellular glutamate accumulation (Haugstad and Langmoen 1996; Hegstad et al. 1996); channel overexpression may protect by blunting glutamate release and glutamatergic excitation, rather than by normalizing metabolism directly. Both vectors also protected against the apoptosis-inducing compound staurosporine, albeit to a lesser extent than for the other insults, likely by reducing the hyperexcitability linked to apoptosis in hippocampal neurons (Ankarcrona et al. 1995; Bredesen 1995). Of note, complete silencing of hippocampal neurons with ROMK1 (Kir1.1) overexpression induces apoptosis (Nadeau et al. 2000), suggesting there is a limit to the protection potential of increased K+ channel expression. SK2 and Kv1.1 differentially alter neuronal physiology, which may explain why Kv1.1 protected over a greater range of glutamate doses while SK2 protected against a greater range of cyanide doses. Variations in protection may be due to differences in magnitude of channel currents, gating properties or cellular localization of the overexpressed channels. Kv and SK channels are relatively conserved; both are a-subunit tetramers with each subunit having six membrane-spanning domains and cytosolic N- and C-termini (Bond et al. 1999). Despite these structural similarities, single channel conductance for SK channels is smaller than for Kv channels (< 100 pS versus > 130 pS) (Farley and Rudy 1988; Bielefeldt et al. 1992). Moreover, calmodulin constitutively binds to the intracellular domain of SK, conferring calcium regulation, but not to Kv subunits (Xia et al. 1998). A larger hyperpolarizing current in the cytosolic membrane might give Kv channels a greater advantage in offsetting the depolarizing actions of KA. Alternatively, differences in gating properties may explain differential protection with Kv channels playing more of a role in shaping the action potential while SK channels influence firing frequency and accommodation (Blatz and Magleby 2 1987; Sah 1996; Hille 2001). It is possible that SK2 overexpression provides greater resistance to cyanide than Kv1.1 because more energy is saved by reducing action potential frequency. Protection against glutamate and KA provided by K+ channel overexpression is consistent with our prediction that



lessening excitability can protect against excitotoxicity. The most simple explanation is that overexpression of K+ channels reduces excessive cell discharge and the damaging processes that ensue. In support, enhancement of PSs in the DG produced by KA is attenuated by Kv1.1 or SK2 overexpression. Similarly, overexpression of KATP channels in hippocampal cells reduces excitability under current clamp (Ehrengruber et al. 1997). While both vectors decreased excitability to the same extent, SK2 was more effective than Kv1.1 at decreasing KA neurotoxicity. In explaining this seeming discrepancy, the PS study was carried out at only a single dose and time point, and the two channels are likely to have differing time courses of efficacy. Kv1.1, Kv1.2 and Kv1.4 are expressed in rat hippocampal neurons with Kv1.1 and Kv1.2 forming heterotetrameric complexes with Kv1.4 (but not each other) at the cell surface. In addition, complexes also occur in organelles such as the endoplasmic reticulum and a perinuclear compartment (Manganas and Trimmer 2000). SK2 is also present in the hippocampus predominantly in dendritic regions of Ammon’s horn and the dentate gyrus (Sailer et al. 2002). If overexpressed SK2 channels translocate to mitochondria, they may be in a better place to abrogate the metabolic failure produced by cyanide. However, endogenous SK2 channels have not been reported in mitochondria (in contrast to KATP and voltage-gated calcium-sensitive K+ channels) (Siemen et al. 1999; Szewczyk and Marban 1999). Delivery of constitutively expressing vectors could mean overexpression of transgenes at a time when there is not yet, or no longer an insult occurring. Therefore, of concern are the current findings that these K+ channels alter normative physiology and behavior and previous work showing effects of other forms of gene transfer on hippocampal function in the absence of an insult (Chard et al. 1995; Dumas et al. 1999). Additionally, the current finding that SK2 overexpression impairs spatial reference memory adds to findings that SK channels are negative regulators of reference memory (Fournier et al. 2001; Stackman et al. 2002). This problem may be circumvented by the development of vectors that can be delivered pre-insult and are induced by the insult (Ozawa et al. 2000). To summarize, overexpression of Kv1.1 or SK2 enhanced survival of hippocampal neurons in vitro and in vivo after excitotoxic insults (and metabolic insults in vitro). Since overexpression of Kv1.1 or SK2 in DG granule cells reduced the neuronal hyperexcitability produced by KA, it is possible they may both provide protection, in part, by reducing activity. Moreover, in the absence of any insult, overexpression of Kv1.1 altered basal excitability, and overexpression of SK2 potentiated medial PP synaptic transmission and impaired spatial memory. Thus, K+ channel gene therapy may be a viable means of preventing clinical neuron death from ischemic insult. However, as the morphological sparing

of neurons we demonstrate here is not equivalent to functional sparing of neurons (Dumas et al. 2000; Dumas and Sapolsky 2001) we are currently testing the effects of K+ channel gene therapy on behavior after excitotoxic insult. Acknowledgements We would like to thank John P. Adelman for the SK2 and Kv1.1 constructs. We acknowledge the technical assistance of Min Chin Lim and Timothy Meier. This research was funded by AHA Fellowship 9920072Y and NSRA NS010879-02 to ALL, NIH Grant AG00563 and the TRDRP program of the State of California to RMS.

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