Disease Modification by Combinatorial Single Vector Gene Therapy: A Preclinical Translational Study in Epilepsy.

Gene therapy has been suggested as a plausible novel approach to achieve seizure control in patients with focal epilepsy that do not adequately respond to pharmacological treatment. We investigated the seizure-suppressant potential of combinatorial neuropeptide Y and Y2 receptor single vector gene therapy based on adeno-associated virus serotype 1 (AAV1) in rats. First, a dose-response study in the systemic kainate-induced acute seizure model was performed, whereby the 1012 genomic particles (gp)/mL titer of the vector was selected as an optimal concentration. Second, an efficacy study was performed in the intrahippocampal kainate chronic model of spontaneous recurrent seizures (SRSs), designed to reflect a likely clinical scenario, with magnetic resonance image (MRI)-guided focal unilateral administration of the vector in the hippocampus during the chronic stage of the disease. The efficacy study demonstrated a favorable outcome of the gene therapy, with a 31% responder rate (more than 50% reduction in SRS frequency) and 13% seizure-freedom rate, whereas no such effects were observed in the control animals. The inter-SRS and SRS cluster intervals were also significantly prolonged in the treated group compared to controls. In addition, the SRS duration was significantly reduced in the treated group but not in the controls. This study establishes the SRS-suppressant ability of the single vector combinatorial neuropeptide Y/Y2 receptor gene therapy in a clinically relevant chronic model of epilepsy.

Unilateral ex vivo gene therapy by GDNF in epileptic rats.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. This neurological disorder is characterized by focal seizures originating in the temporal lobe, often with secondary generalization. A variety of pharmacological treatments exist for patients suffering from focal seizures, but systemically administered drugs offer only symptomatic relief and frequently cause unwanted side effects. Moreover, available drugs are ineffective in one third of the epilepsy patients. Thus, developing more targeted and effective treatment strategies for focal seizures, originating from, e.g., the temporal lobe, is highly warranted. In order to deliver potential anti-epileptic agents directly into the seizure focus we used encapsulated cell biodelivery (ECB), a specific type of ex vivo gene therapy. Specifically, we asked whether unilateral delivery of glial cell line-derived neurotrophic factor (GDNF), exclusively into the epileptic focus, would suppress already established spontaneous recurrent seizures (SRS) in rats. Our results show that GDNF delivered by ECB devices unilaterally into the seizure focus in the hippocampus effectively decreases the number of SRS in epileptic rats. Thus, our study demonstrates that focal unilateral delivery of neurotrophic factors, such as GDNF, using ex vivo gene therapy based on ECB devices could be an effective anti-epileptic strategy providing a bases for the development of a novel, alternative, treatment for focal epilepsies.

Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability.

Neural progenitors in the adult dentate gyrus continuously produce new functional granule cells. Here we used whole-cell patch-clamp recordings to explore whether a pathological environment influences synaptic properties of new granule cells labeled with a GFP-retroviral vector. Rats were exposed to a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus, which gave rise to neuronal death, inflammation, and chronic seizures. Granule cells formed after these stimuli exhibited similar intrinsic membrane properties. However, the new neurons born into the pathological environment differed with respect to synaptic drive and short-term plasticity of both excitatory and inhibitory afferents. The new granule cells formed in the epileptic brain exhibited functional connectivity consistent with reduced excitability. We demonstrate a high degree of plasticity in synaptic inputs to adult-born new neurons, which could act to mitigate pathological brain function.

Chronic BDNF deficiency permanently modifies excitatory synapses in the piriform cortex.

Brain-derived neurotrophic factor (BDNF), aside from its classic neurotrophic role in development and survival of neurons, has been shown to be involved in modification and plasticity of central synapses. In mice with BDNF gene deletion (BDNF+/-), deficits in synaptic transmission are often observed but are reversed readily by administration of BDNF, suggesting its acute effect. In support, blockade of BDNF signaling in wild-type hippocampal slices by TrkB-IgG closely reproduces synaptic alterations observed in BDNF+/- mice. We demonstrate that in BDNF+/- mice, lateral olfactory tract (LOT) synapses exhibit decreased release probability of glutamate, suggested by increased paired-pulse facilitation (PPF) of field excitatory postsynaptic potentials (fEPSPs), as well as by slower blocking rate of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs) by MK-801 in the pyramidal neurons of the piriform cortex. The changes in PPF were not mimicked in wild-type mice by acute blockade of BDNF signaling by TkrB-IgG. These data imply that BDNF deficit during development might lead to chronic changes of excitatory transmission in LOT synapses. Modification of the LOT synapses in BDNF+/- mice was associated with altered inhibitory drive onto the mitral cells from the granule and glomerular neurons, which in turn exhibited decreased renewal rate compared to that in wild-type mice. Taken together, these data suggest that BDNF deficiency can have both acute and more permanent effects on synaptic function, particularly when BDNF signaling is compromised during the early stages of brain development. In the latter case, altered synaptic properties in BDNF+/- mice could be secondary to other complex changes in the brain, e.g., cell survival/proliferation.

Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors.

Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice with the same genetic background and explored anti-epileptic action of NPY in vitro and in vivo. In Y2 (Y2-/-) and Y5 (Y5-/-) receptor knockouts, NPY partially inhibited 0 Mg2+-induced epileptiform activity in hippocampal slices. In contrast, in double knockouts (Y2Y5-/-), NPY had no effect, suggesting that in the hippocampus in vitro both receptors mediate anti-epileptiform action of NPY in an additive manner. Systemic kainate induced more severe seizures in Y5-/- and Y2Y5-/-, but not in Y2-/- mice, as compared to wild-type mice. Moreover, kainate seizures were aggravated by administration of the Y5 antagonist L-152,804 in wild-type mice. In Y5-/- mice, hippocampal kindling progressed faster, and afterdischarge durations were longer in amygdala, but not in hippocampus, as compared to wild-type controls. Taken together, these data suggest that, in mice, both Y2 and Y5 receptors regulate hippocampal seizures in vitro, while activation of Y5 receptors in extra-hippocampal regions reduces generalized seizures in vivo.

Neuropeptide Y Y5 receptors suppress in vitro spontaneous epileptiform bursting in the rat hippocampus.

Neuropeptide Y (NPY) has been implicated in antiepileptic action in different in vivo and in vitro epilepsy models in rats and mice. Both Y2 and Y5 receptors could mediate the seizure-suppressant effect of NPY. However, lack of selective ligands precluded previous studies from conclusively evaluating the role of Y5 receptors in anti-epileptiform action of NPY. In the present study, using the new highly selective Y5 receptor antagonist, CGP71683A, and agonist, [cPP]hPP, we show that the Y5 receptor subtype is centrally involved in NPY-induced suppression of spontaneous epileptiform (interictaform) bursting in the CA3 area of rat hippocampal slices. This novel finding underscores the importance of Y5 receptors as a potential target for future antiepileptic therapy, particularly, for interictal components of temporal lobe epilepsy.

Suppression of limbic motor seizures by electrical stimulation in thalamic reticular nucleus.

Kindling is a model of temporal lobe epilepsy in which repeated electrical stimulations in limbic areas lead to progressive increase of seizure susceptibility, culminating in generalized convulsions and the establishment of a permanent epileptic syndrome. We studied here the effect of stimulations in the thalamic reticular nucleus (TRN) on the development of seizures and hippocampal hyperexcitability in kindling elicited from the ventral hippocampus in rats. Animals given 12 kindling stimulations per day with 30-min intervals for 4 consecutive days developed generalized convulsions on day 4. Stimulations in TRN delivered simultaneously with those in the hippocampus induced marked suppression of seizure generalization. Similarly, the number of generalized seizures and the duration of behavioral convulsions were reduced when rats subjected to 40 kindling stimulations with 5-min intervals during about 3 h were costimulated in the TRN. The anticonvulsant effect of TRN costimulation was detected also when rats were test-stimulated in the hippocampus at 24 h and 2 and 4 weeks after the initial 40 hippocampal stimulations. Our data provide the first evidence that TRN stimulations can act to suppress limbic motor seizures in hippocampal kindling and suggest a new approach for seizure control in temporal lobe epilepsy.

BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion.

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.