Block of neural Kv1.1 potassium channels for neuroinflammatory disease therapyкод для вставкиСкачать
Block of Neural Kv1.1 Potassium Channels for Neuroinflammatory Disease Therapy Evelyne Beraud, PhD,1 Angèle Viola, PhD,2 Imed Regaya, PhD,3 Sylviane Confort-Gouny, PhD,2 Philippe Siaud, PhD,4 Danielle Ibarrola, PhD,2 Yann Le Fur, PhD,2 Jocelyne Barbaria, BS,1 Jean-François Pellissier, MD,5 Jean-Marc Sabatier, PhD,3 Igor Medina, PhD,6 and Patrick J. Cozzone, PhD2 Objective: We asked whether blockade of voltage-gated K⫹ channel Kv1.1, whose altered axonal localization during myelin insult and remyelination may disturb nerve conduction, treats experimental autoimmune encephalomyelitis (EAE). Methods: Electrophysiological, cell proliferation, cytokine secretion, immunohistochemical, clinical, brain magnetic resonance imaging, and spectroscopy studies assessed the effects of a selective blocker of Kv1.1, BgK-F6A, on neurons and immune cells in vitro and on EAE-induced neurological deficits and brain lesions in Lewis rats. Results: BgK-F6A increased the frequency of miniature excitatory postsynaptic currents in neurons and did not affect T-cell activation. EAE was characterized by ventriculomegaly, decreased apparent diffusion coefficient, and decreased (phosphocreatine ⫹ ␤-adenosine triphosphate)/inorganic phosphate ratio. Reduced apparent diffusion coefficient and impaired energy metabolism indicate astrocytic edema. Intracerebroventricularly BgK-F6A–treated rats showed attenuated clinical EAE with unexpectedly reduced ventriculomegaly and preserved apparent diffusion coefficient values and (phosphocreatine ⫹ ␤-adenosine triphosphate)/inorganic phosphate ratio. Thus, under BgK-F6A treatment, brain damage was dramatically reduced and energy metabolism maintained. Interpretation: Kv1.1 blockade may target neurons and astrocytes, and modulate neuronal activity and neural cell volume, which may partly account for the attenuation of the neurological deficits. We propose that Kv1.1 blockade has a broad therapeutic potential in neuroinflammatory diseases (multiple sclerosis, stroke, and trauma). Ann Neurol 2006;60:586 –596 In excitable tissues, voltage-gated K⫹ channels (Kv) serve in repolarizing action potentials and shaping neuronal excitability. Kv1.1 to 1.6 channel ␣-subunits are present in the central nervous system (CNS).1 Kv ␣-subunits are thought to coassemble into heterotetramers in vivo to form Kv channels.2,3 Few subunit combinations have been detected so far: Kv1.1 with Kv1.2 subunits and Kv1.6 with Kv1.1/1.2 subunits.1,3 Heteromultimers of Kv1.1 and Kv1.2 ␣-subunits with the cytoplasmic Kv␤2-subunit are found in the juxtaparanodal regions of myelinated axons throughout the brain.3–5 Kv1.1 and Kv1.2 have a different spatial distribution in altered myelin, which may disturb nerve conduction.5–7 Kv blockers such as aminopyridines (APs) restore conduction to demyelinated axons in vitro and potentiate synaptic transmission in vitro and in vivo.8 4-AP is applied to demyelinating disease such as multiple sclerosis (MS) and spinal cord injury treatment.7,8 MS is characterized by inflammation, demyelination, and axonal damage.9 The use of 4-AP is limited by narrow toxic-to-therapeutic ratio, which may result from AP blocking a vast array of Kv. More selective Kv blockers may serve as potential symptomatic therapy for CNS diseases. Attention has been focused From the 1Service d’Immunologie, Faculté de Médecine, Université de la Méditerranée; 2Centre de Résonance Magnétique Biologique et Médicale, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6612, Faculté de Médecine, Université de la Méditerranée; 3Laboratoire d’Ingénierie des Protéines, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6560; Institut Fédératif de Recherche Jean Roche; 4Laboratoire d’Otologie, Equipe Propre Institut National de la Sante et de la Recherche Médicale 19902, Institut Fédératif de Recherche Jean Roche; 5Laboratoire de Biopathologie Nerveuse et Musculaire, Faculté de Médecine, Université de la Méditerranée; and 6Institut de Neurobiologie de la Méditerranée, Institut National de la Santé et de la Recherche Médicale U29, Marseille, France. Current address for Dr Beraud: Institut National de la Santé et de la Recherche Médicale Unité 777, Faculté de Médecine, Université de la Méditerranée, Marseille, France. Current address for Drs Regaya and Sabatier: Equipe de Recherche Technologique 62, Ingénierie des Peptides à visée Thérapeutique, Faculté de Médecine Nord, Université de la Méditerranée, Marseille, France. This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat Received May 17, 2006, and in revised form Sep 8. Accepted for publication Sep 12, 2006. E.B. and A.V. contributed equally to this work. 586 Published online Oct 16, 2006, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21007 Address correspondence to Dr Beraud, Institut National de la Sante et de la Recherche Médicale Unité Mixte de Recherche, 777, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. E-mail: email@example.com © 2006 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services on blockade of Kv1.3 because this homotetrameric channel, together with the calcium-activated K⫹ channel (IKCa3.1), regulates Ca2⫹ signaling by controlling membrane potential in T lymphocytes, and thus is viewed as a potential therapeutic target for novel immunomodulators.10 However, current immunosuppressive or immunomodulatory treatments have little effect in patients with progressive MS.11,12 Adjunct therapies are lacking, given the complexity of MS. Here, we target neural Kv in experimental autoimmune encephalomyelitis (EAE), a largely accepted model of MS,13 by a blocker highly selective for Kv1.1. BgK is a peptide binding with similar affinity to Kv1.1, Kv1.2, Kv1.3,14 and Kv1.6. but not to Kv1.4 and Kv1.5 channels.15 The selectivity of BgK for Kv1 can be altered by Phe6 to Ala substitution, which reduces the affinity of BgK for homomultimeric Kv1.2 and Kv1.3 without affecting the affinity for homomultimeric Kv1.1 (with a 50% inhibiting concentration [IC50] of 0.7nM).14 We synthesized this analogue, BgK-F6A, and studied its electrophysiological effects on hippocampal neurons in culture. We also ascertained its lack of effects on Kv1.3⫹ T-cell activation. Then, we examined the impact of intracerebroventricular (ICV) infusion of BgK-F6A on clinical EAE. Last, we identified structural and metabolic markers related to EAE for assessing cerebral effects of BgK-F6A, using a multimodal magnetic resonance (MR) approach. Because phospholipids and bioenergetics are altered not only in MS plaques but also in normal-appearing white matter,16,17 we monitored cerebral metabolism using phosphorus magnetic resonance spectroscopy (MRS). We report that BgK-F6A markedly attenuates clinical EAE progression and neurological outcomes, including brain damage and energy metabolism. Materials and Methods Animals Animal studies followed the guidelines in France and were approved by the local Ethics Committee. Female Lewis rats (6 and 12 weeks old) were from Charles River-Iffa Credo, Breeding Laboratories (L’Arbresle, France). Reagents Myelin basic protein (MBP) from frozen spinal cords of guinea pigs18 (Harlan, Gannat, France) was purified by C18 reverse-phase high-pressure liquid chromatography with a Millipore/Waters Associates system (Milford, DE). BgK-F6A was synthesized as described elsewhere.19 The peptide was characterized by amino acid analysis, Edman sequencing, and mass spectrometry. Cells and T-Cell Line The MBP-T cell line called PAS was established from MBPprimed Lewis rat lymph nodes and characterized as cytotoxic, major histocompatibility complex class II–restricted CD4⫹ T cells that are encephalitogenic in vivo.18 They pro- duce limited demyelination. For antigen activation, PAS T cells (3 ⫻ 105/ml) were incubated 2 days with 10g/ml MBP and 15 ⫻ 106/ml syngeneic-irradiated (2,500rad) thymocytes as antigen-presenting cells in RPMI-1640 Dutch modification medium supplemented with 1% syngeneic rat serum and additives (stimulation medium). Blasts were expanded in interleukin-2 growth medium supplemented with 10% fetal calf serum. Experimental Autoimmune Encephalomyelitis Models ADOPTIVELY TRANSFERRED MUNE ENCEPHALOMYELITIS. EXPERIMENTAL AUTOIM- The MBP-activated T cells were intraperitoneally injected in 1ml phosphate-buffered saline (1–5 ⫻ 106 blasts/rat). ACTIVELY INDUCED EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS. Rats were immunized by subcutane- ous injection at the tail base with MBP emulsified in incomplete Freund adjuvant from Sigma-Aldrich (Saint Quentin Fallavier, France) supplemented with H37Ra Mycobacterium tuberculosis (Difco, Detroit, MI). Each animal received 200l emulsion containing 25g MBP and 400g mycobacteria. CLINICAL EVALUATION. Disease severity was scored from 0 to 6 with 0.5 increments for intermediate findings. Rats showing no signs of EAE and no weight loss were excluded from the studies. Statistics were done with Mann–Whitney U test. Significance was set at values of p ⬍ 0.05. DATA ANALYSIS. Drug Delivery In our previous studies demonstrating the immunoregulatory properties of kaliotoxin (KTX) (a blocker of Kv1.1 and Kv1.3)20 and ShK (a blocker of Kv1.1, Kv1.3, and Ca2⫹activated K⫹ channel [KCa3.1]),21 systemic injections maintained channel blockade in vivo from the day of the adoptive transfer and/or during the disease. Here, the drug was infused continuously to ensure optimal delivery of BgK-F6A in situ. For ICV administration, a cannula was stereotaxically implanted into the right lateral ventricle under subcutaneous anesthesia with ketamine (400l/kg) and medetomidine (150l/kg) (coordinates: anterior ⫽ 0.8mm, lateral ⫽ 1.4mm, and ventral ⫽ ⫺4.2mm, relative to bregma).22 The metallic cannula (Alzet brain infusion kit II; Durect Corporation, Cupertino, CA) was imbedded into the skull with dental cement and connected by a catheter to an osmotic pump (model 2001: 1.0l/hr, 7 days; model 2002: 0.5l/hr, 14 days, as indicated) implanted subcutaneously on the back. Teflon and silica cannulas (Bilaney, Dusseldorf, Germany) were used for MR studies. In Vivo Magnetic Resonance Protocol The rats were explored on a 4.7-Tesla horizontal Bruker AVANCE Biospec MR system (47/30) (Bruker, Karlsruhe, Germany). Animals were anesthetized with isoflurane 1.5 to 2% and placed in a cradle equipped with a stereotaxic holder and a heating system to maintain the body temperature at 36 ⫾ 1°C and a pressure probe to monitor respiration. Beraud et al: Neural Kv1.1 Blockade Improves EAE 587 Table 1. BgK-F6A Intracerebrovascular Treatment Improves Adoptively Transferred Experimental Autoimmune Encephalomyelitis Individual Maximal Clinical EAE Scoresa Treatmentb n None Saline BgK-F6A 2 10 5 0 1.5 2 1 1 3 3 4 5 6 Mortality Mean of Maximum Clinical Scores ⫾ SD 2 9 1 100% 90% 20% 6 5.6 ⫾ 1.3 2.7 ⫾ 1.8 pc ⬍0.01 The severity of the disease was scored on a scale of 0 to 6 (0 ⫽ no clinical signs; 1.0 ⫽ limp tail; 2.0 ⫽ mild paraparesis and ataxia; 3.0 ⫽ moderate paraparesis; 4.0 ⫽ complete hind limb paralysis; 5.0 ⫽ paralysis ⫹ incontinence; 5.5 ⫽ tetraplegia; 6.0 ⫽ moribund or death). The data represent the number of rats with their maximal clinical score for each group. a b Determination of optimal drug dose: The 50% lethal dose (LD50) of BgK-F6A injected intracerebrovascularly (ICV) into 20g mice was 85ng, 4-fold the dose of kaliotoxin.40 The quantities of blockers to be delivered by the osmotic pumps were first determined on the basis of daily ICV injection results. With that mode of continuous administration in preliminary experiments, even the lowest dose was toxic, and thus was reduced. ICV cannula and subcutaneous osmotic pumps containing BgK-F6A (20-25ng/0.5l/hr) or saline (NaCl 0.15M) were implanted in female Lewis rats. Three to 5 days later, rats received encephalitogenic activated myelin basic protein T-line PAS lymphocytes intraperitoneally. c Mann–Whitney U test. Mean differences between groups were considered significant at values of p ⬍ 0.05. EAE ⫽ experimental autoimmune encephalomyelitis; SD ⫽ standard deviation. Geometric parameters for multislice T1- and T2-weighted images were as follows: 15 slices 1mm in thickness; interslice distance, 1mm; matrix, 2562; and field of view, 302mm2. Axial T1- and T2weighted images were acquired using a spin-echo sequence (T1-weighted images: TE ⫽ 15 milliseconds; TR ⫽ 630.61 milliseconds; T2-weighted images: TE ⫽ 40 milliseconds; TR ⫽ 2,500 milliseconds). T1-weighted images were collected before and after gadolinium-diethylenetriamine pentaacetic acid injection (1ml/kg; Schering-Plough, LevalloisPerret, France). Multislice diffusion-weighted spin-echo echo planar imaging mapped the apparent diffusion coefficient (ADC) using 15 contiguous slices and 5 increasing values of b-factor.23 BRAIN MAGNETIC RESONANCE IMAGING. 31 PMRS was performed using a homemade surface coil (1cm diameter) tuned to 31P (81.184MHz). The spectra were acquired as described elsewhere.23 BRAIN MAGNETIC RESONANCE SPECTROSCOPY. Magnetic Resonance Data Processing MR data were processed under IDL environment (Interactive Data Language Research System, Boulder, CO). MAGNETIC RESONANCE IMAGING DATA PROCESSING. Cerebral and ventricular (lateral and third ventricles) volumes and ADC maps were obtained as reported elsewhere.23 Regional ADC values were evaluated as an average of pixel values in the cortex and the striatum. 31 P MAGNETIC RESONANCE SPECTROSCOPIC DATA PROCESSING. In vivo brain 31P-MRS gives access to the signals of adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi), and phosphomonoesters (PME). In addition, a broad “bump” is observed, due to the phosphorus contained in the skull. Spectra were processed as described elsewhere23 and referenced to PCr for chemical shift [⫺2.45 ppm]. Resonance signals from PCr, Pi, and ␣-, ␤-, and ␥-ATP were integrated using the AMARES-MRUI FORTRAN line-fitting procedure. The bone signal was sub- 588 Annals of Neurology Vol 60 No 5 November 2006 tracted by deconvolution. The chemical shift between Pi and PCr was used to calculate brain intracellular pH.23 STATISTICAL ANALYSIS. All MR results are mean values ⫾ standard error. Data were compared using Student’s t test. Significance was set at p ⬍ 0.05. Results BgK-F6A has the same high affinity for Kv1.1 as the parental peptide (IC50: 0.72nM) but decreased affinities for Kv1.2 (IC50: 400nM) and Kv1.3 (IC50: 800nM). These IC50 values, determined by whole-cell patch-clamp for rat Kv expressed in oocytes, were thus 500- to 1,000-fold higher than IC50 for Kv1.1.14 Therapy of Adoptively Transferred Experimental Autoimmune Encephalomyelitis by Brain Infusion of a Neural Kv Blocker BgK-F6A improved the neurological status in four of five rats capable of walking (Table 1). In striking contrast, 9 of 10 saline-treated control rats (90%) died of EAE. Lethality was 100% in the other control group without ICV implantation. Two rats, one control rat without ICV implantation and one treated with BgKF6A, were excluded from the studies concerning the therapy of adoptively transferred EAE because they did not show any signs of EAE and weight loss. BgK-F6A– protected rats presented unusual but nonaggressive reactivity by jumping away with bursts of energy as the hand of the investigator approached them. No other side effect was observed, apart from a few seizures in two rats. The unprotected rat was calm, as were the control rats. Given BgK-F6A ability to improve EAE, we conducted ICV treatment trials in EAE actively induced by MBP. This model induces less lethality, and thus allows kinetic studies by MRI and MRS. Blood–brain barrier (BBB) lesions were detected on contrast-enhanced, T1-weighted MRI with gadoliniumdiethylenetriamine pentaacetic acid, which showed increased signal intensity in periventricular regions and in ventricles in saline-treated rats, the latter being a sign of ventriculitis (Fig 3).24 Compared with saline-treated rats, ICV BgK-F6A–treated rats showed no reduction in signal enhancement. Fig 1. BgK-F6A intracerebroventricular (ICV) treatment improves actively induced experimental autoimmune encephalomyelitis (EAE). Lewis rats were immunized with MBP⫹ complete Freund’s adjuvant (CFA) at day 0. Four and 5 days later, ICV cannula and osmotic pumps, delivering 0.5l/hr during 14 to 18 days, were implanted in these rats. Eight rats were treated by BgK-F6A (open diamonds; 6.5 and 15ng/ 0.5l/hr; mean maximal score, 2.37 ⫾ 0.8) and 10 rats by saline (triangles; mean maximal score, 5.15 ⫾ 0.4) (2 experiments). *p ⬍ 0.01 compared with saline group. These animals were submitted to magnetic resonance imaging and magnetic resonance spectroscopy investigations. MBP ⫽ myelin basic protein. Therapy of Actively Induced Experimental Autoimmune Encephalomyelitis by Brain Infusion of a Neural Kv Blocker All eight rats receiving BgK-F6A improved drastically (Fig 1). These animals kept on walking and grooming. In contrast, the 10 saline-treated rats developed full paralysis. Moreover, BgK-F6A–treated rats presented unusual reactivity, as described earlier, except in one rat with a score of 3. No side effect was observed besides epileptic fits in one rat (score of 2). Results were similar when BgK-F6A was injected either at 6.5 or at 15ng/0.5l. Hence, BgK-F6A greatly attenuated the progression of clinical EAE, showing that Kv1.1 blockade improves the symptoms. We therefore investigated these rats to determine whether the neurological beneficial effects of BgK-F6A treatment could be assessed by MRI and MRS. Treatment with BgK-F6A Prevents Ventriculomegaly but Does Not Decrease Blood–Brain Barrier Lesions in Experimental Autoimmune Encephalomyelitis Rats Intracerebroventricular saline-treated rats presented major ventriculomegaly visible on T2-weighted images (Fig 2A). Ventricle enlargement was significant but no brain swelling was detected (Table 2). Ventricle size returned to normal during EAE recovery (data not shown). In contrast, BgK-F6A–treated rats did not show ventricle enlargement (see Fig 2B and Table 2). Treatment with BgK-F6A Maintains Normal Brain Apparent Diffusion Coefficient in Rats Induced for Experimental Autoimmune Encephalomyelitis The ADC maps (Fig 4A) showed reduced ADC values in the whole brain, with significantly decreased ADC values in the striatum (see Fig 4B) and in the parietal cortex (see Fig 4C). In contrast, rats receiving BgKF6A displayed normal ADC values in the striatum and in the cortex (see Figs 4D, E). MRI exploration of three healthy rats receiving BgK-F6A showed no brain lesion or changes in ADC values (data not shown). The ADC values of healthy control rats agreed with previous results.25 Treatment with BgK-F6A Preserves Normal Brain Energy Metabolism in Rats Induced for Experimental Autoimmune Encephalomyelitis Brain 31P spectra showed significantly reduced ratios of phosphocreatine to inorganic phosphate (PCr/Pi), ␤-ATP/Pi, and (PCr ⫹ ␤-ATP)/Pi in saline-treated rats (Figs 5A, B; Table 3). Partial metabolic recovery was observed during EAE clinical recovery (see Fig 5C). Remarkably, EAE-induced rats receiving BgKF6A showed no alteration of their cerebral energy metabolism (see Figs 5D, E; see Table 3), like the three healthy control rats receiving BgK-F6A (data not shown). Discussion Our data provide the first evidence that a blocker of neural Kv1.1, BgK-F6A, attenuates clinical EAE, reduces cerebral injury, and preserves brain bioenergetics. Immune Function–Independent Action of BgK-F6A BgK-F6A exhibits 500- to 1,000-fold higher selectivity for Kv1.1 than Kv1.2 and Kv1.3. We confirmed its decreased affinity for Kv1.3, because BgK-F6A had no effects on T-cell activation (see Supplementary material). Kv1.3 is the main channel for the outward current of activated T cells,26,27 B cells,28 macrophages,29 and microglia30 in humans and rats, and dendrocytes in mice.31 No functional expression of Kv1.1 has been reported. Thus, T-cell Kv1.3 is a potential therapeutic target for novel immunomodulators. We previously presented the first proof-of-concept for preventing and treating a CNS autoimmune disease by immunomodulation with a Kv1.3 blocker (kaliotoxin).20 Other Beraud et al: Neural Kv1.1 Blockade Improves EAE 589 Fig 2. Typical axial brain T2-weighted images show reduced ventriculomegaly in intracerebroventricular (ICV) BgK-F6A–treated rats. (A) Images from an ICV saline-treated rat before and during experimental autoimmune encephalomyelitis (EAE) showing pronounced ventriculomegaly during EAE (arrows). (B) Images from an ICV BgK-F6A–treated rat before and during EAE. Only a slight ventricular dilatation is detected during EAE (arrows). C ⫽ cannula. Scale bar ⫽ 2mm. blockers of Kv1.3 [ShK, ShK-Dap, and ShK(L5)] were shown to act as immunomodulators and to improve acute EAE.21,32 Because most of these blockers can also theoretically act on the CNS because of their additional selectivity for Kv1.1, the EAE improvement 590 Annals of Neurology Vol 60 No 5 November 2006 could result from immunomodulatory abilities combined with beneficial neural effects. Here, we analyzed the consequences of Kv1.1 blockade only, and its implication in EAE improvement, excluding immunomodulation via Kv1.3 blockade. Table 2. Brain Volumetry Saline-Treated Rats (n ⫽ 6) Characteristics Mean brain volume ⫾ SE (mm3) Mean ventricle volume ⫾ SE (mm3) Mean ventricle volume/brain volume ⫾ SE Mean parenchymal fractiond ⫾ SE a Before EAE 3,273 ⫾ 35 During EAE 3,234 ⫾ 58 BgK-F6A–Treated Rats (n ⫽ 5) Before EAE 3,207 ⫾ 33 During EAE 3,214 ⫾ 26 90 ⫾ 12 150 ⫾ 16a,b 70 ⫾ 10 74 ⫾ 8 0.028 ⫾ 0.004 0.046 ⫾ 0.004a,c 0.022 ⫾ 0.005 0.023 ⫾ 0.005 0.970 ⫾ 0.004 0.950 ⫾ 0.004a,b 0.980 ⫾ 0.003 0.970 ⫾ 0.004 Student’s t test. Mean differences between data series were considered significant at values of p ⬍ 0.05. bp ⬍ 0.05; cp ⬍ 0.01. Parenchymal fraction: (brain volume ⫺ ventricle volume)/brain volume. EAE ⫽ experimental autoimmune encephalomyelitis; SE ⫽ standard error. d Role of BgK-F6A in Neuronal Excitability Cultured hippocampal neurons show a developmental profile, subcellular localization, and functionality of Kv1.1 similar to those described in situ.33 BgK-F6A Fig 3. Evidence of blood–brain barrier (BBB) lesions and vascular damage in rats with experimental autoimmune encephalomyelitis (EAE). (A) Typical axial contrast-enhanced T1-weighted magnetic resonance images (MRIs) from an intracerebroventricular (ICV) saline-treated rat and an ICV BgK-F6A–treated rat before EAE. (B) Contrast-enhanced T1weighted MRIs from the ICV saline-treated rat and the ICV BgK-F6A–treated rat during EAE. (C) Contrast-enhanced T1-weighted MRIs from the ICV saline-treated rat and the ICV BgK-F6A–treated rat during EAE recovery. Arrows indicate areas of contrast enhancement. Scale bar ⫽ 2mm. efficiently acted on neurons as shown by the increased frequency of the miniature AMPA excitatory postsynaptic currents (see Supplementary material). Our in vitro data suggest that BgK-F6A– and DTX-sensitive K⫹ channels participate in the setting of the membrane potential of presynaptic terminals. Blocking these channels increases the spontaneous synaptic release of glutamate, possibly leading to increased neuronal excitability. Reduction of Neurological Disability under BgK-F6A Treatment: Lack of Correlation between Clinical Experimental Autoimmune Encephalomyelitis Attenuation and Stress and Impairment of Inflammatory Cell Infiltration in the Central Nervous System Adoptively transferred EAE and actively induced EAE in Lewis rats are mainly acute diseases, characterized by an intense and transient inflammation, which occurs largely independently of demyelination and probably pertains to conduction block.34 In both models, in situ BgK-F6A administration improved the clinical course in a dose-dependent manner. Only the rats receiving the highest doses of BgK-F6A developed a few epileptic fits, thus ruling out that stress due to epilepsy might have improved EAE per se. Also, blood cortisol concentration in rats successfully treated by Kv1.1 blockers and presenting the “jumping behavior” was not higher than that of rats inefficiently treated or rats treated by saline (data not shown). This suggests that the “twitchiness” does not cause noticeable steroid-induced stress. Histopathological data of rats clinically improved by BgK-F6A treatment showed typical EAE cell infiltration, which indicates that BgK-F6A did not function by inhibiting immune cell migration into the CNS (see Supplementary material). Beraud et al: Neural Kv1.1 Blockade Improves EAE 591 Fig 4. Apparent diffusion coefficient (ADC) maps show the disappearance of cellular edema in intracerebroventricular (ICV) BgKF6A–treated rats. Representative ADC maps from an ICV saline-treated rat before and during experimental autoimmune encephalomyelitis (EAE). Scale bar ⫽ 1mm. ADC values in the striatum (B) and cortex (C) obtained from diffusion maps for ICV salinetreated rats before (white bars; n ⫽ 6) and during EAE (gray bars; n ⫽ 8). The ADC maps showed decreased ADC values in the striatum (B: before EAE, 0.833 ⫾ 0.033 ⫻ 10⫺3mm2/s; during EAE, 0.789 ⫾ 0.039 ⫻ 10⫺3mm2/s) and in the parietal cortex (C: before EAE, 0.783 ⫾ 0.031 ⫻ 10⫺3mm2/s; during EAE, 0.688 ⫾ 0.02 ⫻ 10⫺3mm2/s). ADC values in the striatum (D) and cortex (E) for ICV BgK-F6A–treated rats before (n ⫽ 7) and during EAE (n ⫽ 8). The ADC maps showed no difference before and during EAE in BgK-F6A–treated rats. Scale bar ⫽ 2mm. Neurons and Astrocytes as In Vivo Potential Targets for Kv1.1 Blockade Acute EAE triggers no or little demyelination, indicating that targets unrelated to myelin or oligodendrocytes may contribute to pathogenesis,35 such as injured axons, implicated during early inflammation in MS and EAE.36,37 We report here on axonal damage during monophasic EAE, quantitatively assessed by immu- 592 Annals of Neurology Vol 60 No 5 November 2006 noreactivity for abnormally dephosphorylated neurofilaments on longitudinal sections of spinal cords (see Supplementary material). Axonal damage may result from compromised energy supply possibly due to inflammatory mediators such as nitric oxide, which initiates a cascade of Na⫹ and Ca2⫹ accumulation and/or due to partial ischemia caused by edema and the infiltration of immune cells.38 Thus, one could expect Fig 5. In vivo cerebral 31P magnetic resonance (31P-MR) spectra evidence of the preservation of brain energy metabolism on BgKF6A treatment in rats. (A–C) Representative original and deconvoluted 31P-MR spectra from an intracerebroventricular (ICV) saline-treated rat before (A), during (B), and after recovery (C) from experimental autoimmune encephalomyelitis (EAE), respectively. (D–F) Representative original and deconvoluted 31P-MR spectra from an ICV BgK-F6A–treated rat before (D), during (E), and after recovery (F) from EAE. The spectra were deconvoluted for signal integration. The signal due to bones (broad bump) was subtracted by deconvolution. Peak assignments: ␣-, ␤-, and ␥-adenosine triphosphate; PCr ⫽ phosphocreatine; Pi ⫽ inorganic phosphate; PME ⫽ phosphomonoesters. Kv1.1 blockade to attenuate the consequences of the disruption of Na⫹ homeostasis, and thereby to diminish axonal injury. Yet, we found a similar extent of axonal injury in both groups of rats induced for EAE, regardless of treatment. However, this finding obtained during EAE recovery might not reflect axonal insult at the peak of the disease in saline-treated rats. Investigations in correlation with clinical symptoms should assess the potential effects of BgK-F6A on axonal injury. Also, as demonstrated in vitro by increased neuronal excitability, BgK-F6A may help restore or enhance axon functionality of injured, “mildly” injured (appearing normal on staining), and normal axons under inflammation process. This is corroborated by the in vivo effects of 4-AP, independent of demyelination, which potentiates synaptic transmission.39 Increased synaptic efficiency in rats after ICV injection of kaliotoxin, a blocker of Kv1.1 and Kv1.3, may also improve associative learning.40 Under mitotic activity, astrocytes have outward K⫹ Table 3. Brain Metabolite Analysis of Rats Determined from In Vivo Brain Metabolite ratiosa (mean ⫾ SE) PCr/␤-ATP PCr/Pi ␤-ATP/Pi (PCr ⫹ ␤-ATP)/Pi pH a Saline-Treated Rats (n ⫽ 12) 31 P Magnetic Resonance Spectroscopy BgK-F6A–Treated Rats (n ⫽ 8) Before EAE During EAE Before EAE During EAE 2.07 ⫾ 0.19 15.12 ⫾ 2.03 8.19 ⫾ 1.33 23.31 ⫾ 3.20 7.06 ⫾ 0.02 2.11 ⫾ 0.09 10.44 ⫾ 0.81a,b 5.08 ⫾ 0.44a,b 15.52 ⫾ 1.21a,c 7.08 ⫾ 0.04 1.83 ⫾ 0.10 13.38 ⫾ 1.65 7.23 ⫾ 0.73 20.61 ⫾ 2.35 7.15 ⫾ 0.08 2.487 ⫾ 0.330 14.36 ⫾ 1.11 6.16 ⫾ 0.61 20.53 ⫾ 1.33 7.12 ⫾ 0.08 Student’s t test. Mean differences between data series were considered significant at values of p ⬍ 0.05. bp ⬍ 0.05; cp ⬍ 0.01. SE ⫽ standard error; EAE ⫽ experimental autoimmune encephalomyelitis; PCr ⫽ phosphocreatine; ATP ⫽ adenosine triphosphate; Pi ⫽ inorganic phosphate (Pi). Beraud et al: Neural Kv1.1 Blockade Improves EAE 593 currents, including Kv1.141; thus, they might be additional targets of BgK-F6A. At the end of the trials, counts of reactive astrocytes were similar in EAE rats treated with saline or BgK-F6A, which suggests that BgK-F6A had no marked effects on astrogliosis at recovery. Astrocytes participate in local immunomodulation42 and protect neurons against excitotoxins and oxidants. Moreover, their impairment secondary to energy depletion may contribute to neuronal injury during ischemia.43 However, as in numerous degenerative conditions, EAE pathology involves early active contribution from astrocytes.44 Astrocyte functions most likely vary with disease progression, and the potential effects of Kv1.1 blockade on them should be investigated. A Novel Characterization of Experimental Autoimmune Encephalomyelitis Model and Insight into Pathological Processes Demonstrated by BgK-F6A Effects Our MR data provide an innovative characterization of EAE highlighting decreased ADC values and impaired brain energy metabolism, and they show the effects of Kv1.1 blockade effects demonstrating markedly reduced cerebral injury and preserved bioenergetics. Ventriculomegaly was reported in EAE,24 but no mechanism was proposed. In patients with MS, with severe disability, ventriculomegaly correlates with brain atrophy.45 We detected only slight atrophy in salinetreated rats with EAE, which could not account for the pronounced ventriculomegaly. It may result from vasogenic edema, a consequence of BBB leakage. Yet, the BgK-F6A–treated rats still showed BBB leakage and cell infiltration but no marked ventriculomegaly. Ventriculomegaly may also result from increased cerebrospinal fluid secretion. Water homeostasis in brain is maintained by regulatory processes involving aquaporins (AQPs). AQP1 is found on epithelial cells in the choroid plexus, whereas AQP4, AQP5, and AQP9 are localized on perivascular astrocytes and ependymal cells.46 Perivascular AQP4 could serve as an influx route of water in conditions favoring edema formation.47 Interestingly, enhanced cerebral expression of AQP4 was found in MS.48 Moreover, the ion concentration in brain is regulated independently of plasma levels by active transport, assisted by astrocytes, across choroid plexus epithelium and cerebral capillary endothelium. Given that Kv1.1 and Kv1.3 contribute largely to Kv conductance in the choroid plexus cells of rat brain in the ventricles,49 our MRI data indicate that epithelial cells of choroid plexus and astrocytes may serve in modulating water flux under Kv1.1 blockade. Cerebral ADC values were decreased in salinetreated rats. In MS, however, ADC values in lesions are increased due to tissue loss.50 Yet, the optic nerves of MS patients with acute neuritis show lower ADC val- 594 Annals of Neurology Vol 60 No 5 November 2006 ues than those with chronic neuritis,51 suggesting that acute inflammation lowered ADC at an early stage of the disease. The same ADC reduction was observed in EAE without any evidence of lesion.52,53 These ADC changes were accounted for by cell infiltration. Here, we show that EAE is associated with reduced ADC concomitant with cell infiltration, whereas BgK-F6A administration during EAE preserves normal ADC values despite persistent cell infiltration. Thus, these results indicate cellular edema.54 It is predominantly due to astrocyte swelling,55 observed in EAE.56 Astrocyte swelling impairs astrocyte function and neuronal activity, due to the release of free radicals or excitatory amino acids in a reduced extracellular space.57 Cellular edema, which is associated with impaired energy metabolism,57 may result from anoxic depolarization after the failure of Na⫹/K⫹ ATPases to maintain membrane potential on ATP depletion. Cerebral high-energy phosphate metabolites are strongly reduced in MS.16,17 As in MS, we found impaired energetic metabolism characterized by lower PCr/Pi and ␤-ATP/Pi ratios affecting the whole brain together with lower ADC values in saline-treated rats with EAE. The lower PCr level reflects an increased ATP demand. Interestingly, MRS studies of swollen astrocytes showed large reductions in PCr and ATP concentrations.58 Moreover, immunostimulation of astrocytes results in ATP depletion,59 which may diminish their neuroprotective action60 and alter signaling with neurons.61 Contributions of Astrocytic Edema and Energy Failure to Experimental Autoimmune Encephalomyelitis Symptoms and BgK-F6A Countereffects Brain energy failure, a potential disease mechanism, may be related to axons and astrocytes, and in particular, to a potentially impaired genetic ability of mitochondria to synthesize ATP in neurons of patients with MS.62 Yet, no direct correlation has been established between mitochondrial dysfunction and reduced ATP level in MS. It is unclear at which level Bgk-F6A acts to maintain the energy metabolism. Cellular edema may contribute, like vasogenic edema, to neurological deficits in EAE. Cellular edema reduction may, therefore, improve clinical symptoms. In that respect, neuroprotective therapies targeting K⫹ channels have proved efficient in animal models of cerebral stroke, a condition with large cellular edema.63 Thus, mechanisms associated with the beneficial effects of BgK-F6A involve differential actions on neural cells and may be independent of a direct action on immune cells. This notion is supported by the persistence of inflammation (BBB lesions and cell infiltrations) in rats treated with BgK-F6A and the lack of in vitro reduction of T-cell activation. Yet, one cannot exclude that BgK-F6A has indirect effects on immune cells, particularly because neurons also play a pivotal role in downregulating local immune response.64 Neuroprotective therapies involve AMPA antagonists35; cannabinoids, which stimulate receptors expressed throughout the CNS37; and sodium channel blockers phenytoin65 and flecainide,38 which protect axons from degeneration in EAE. Kv1-blockade therapy in CNS may have an advantage because it combines abilities to reduce edema, to preserve or to quickly restore brain energetics, and most likely to increase neuronal excitability as triggered by APs, which are far less selective than BgK-F6A. One limitation of this study is the mode of drug administration. Complexation of BgK-F6A with certain cyclodextrins may be a promising delivery system. Kv1.3 is highly expressed on inflammatory infiltrates in MS brain.66 Thus, encapsulated Kv1.1/1.3 blockers might be designed for a combined and local action in various MS patterns, even in those not responding to current immunosuppressant and immunomodulator therapies. Here, we provide preclinical evidence suggesting that Kv1.1 blockers, complementary to other therapies, may treat neuroinflammatory diseases such as MS, stroke, and trauma. 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