Hippocampal GAD67 Transduction Using rAAV8 Regulates Epileptogenesis in EL Mice

Abstract

Gene therapy has been employed as a therapeutic approach for intractable focal epilepsies. Considering the potential of focal GABAergic neuromodulation in regulating epileptogenesis, the GABA-producing enzyme, γ-aminobutyric acid decarboxylase 67 (GAD67), is highly suitable for epilepsy therapy. The EL/Suz (EL) mouse is a model of multifactorial temporal lobe epilepsy. In the present study, we examined focal gene transduction in epileptic EL mice using recombinant adeno-associated virus serotype 8 (rAAV8) expressing human GAD67 to enhance GABA-mediated neural inhibition. Eight-week-old mice were bilaterally injected with rAAV8-GFP or rAAV8-GAD67 in the hippocampal CA3 region. After four weeks, the GAD67-transduced EL mice, but not the rAAV-GFP-treated EL mice, exhibited a significant reduction in seizure generation. The GAD67-mediated depression became stable after 14 weeks. The excitability of the CA3 region was markedly reduced in the GAD67-transduced EL mice, consistent with the results of the Ca2+ imaging using hippocampal slices. In addition, downregulation of c-Fos expression was observed in GAD67-transduced hippocampi. Our findings showed that rAAV8-GAD67 induced significant changes in the GABAergic system in the EL hippocampus. Thus, rAAV8-mediated GAD67 gene transfer is a promising therapeutic strategy for the treatment of epilepsies.

Global brain delivery of neuroligin 2 gene ameliorates seizures in a mouse model of epilepsy

“…Brain samples for immunohistochemistry were prepared as described previously, 18 Briefly, after deeply anesthetized with pentobarbital sodium at 20 weeks of age following the last evaluation, each mouse was intracardially perfused with saline, followed by 4% paraformaldehyde in 0.05 m PBS (pH 7.4). The brain was dissected out immediately, immersed in a fixative for a half‐day at 4°C, and transferred into a 15% sucrose solution in PBS (pH 7.4) at 4°C.…”

“…Gene therapy for epilepsy has been performed as preclinical studies on rodent models chiefly targeting focal epilepsy such as temporal lobe epilepsy by local injection of viral vectors. Local induction of preprodynorphin, glutamate‐gated chrolide channel, neuropeptide Y and Y2, voltage‐gated potassium channel, and collybistin, etc., have demonstrated positive outcomes of vector‐mediated gene therapy for epilepsy 14–19 . We also have demonstrated that AAV8 vector‐mediated gene delivery of glutamic acid decarboxylase ( GAD ), a rate‐limiting enzyme for synthesizing inhibitory neuronal transmitter GABA, into the hippocampus of epileptic (EL) mice reduces seizure frequency and strength, but not seizure duration of tonic–clonic convulsion 18 .…”

“…Local induction of preprodynorphin, glutamate‐gated chrolide channel, neuropeptide Y and Y2, voltage‐gated potassium channel, and collybistin, etc., have demonstrated positive outcomes of vector‐mediated gene therapy for epilepsy 14–19 . We also have demonstrated that AAV8 vector‐mediated gene delivery of glutamic acid decarboxylase ( GAD ), a rate‐limiting enzyme for synthesizing inhibitory neuronal transmitter GABA, into the hippocampus of epileptic (EL) mice reduces seizure frequency and strength, but not seizure duration of tonic–clonic convulsion 18 . Because seizures develop across the brain in many forms of epilepsy, global delivery of the vectors is necessary to tackle such primary or secondary generalized seizures.…”

The Role of Phospholipase C in GABAergic Inhibition and Its Relevance to Epilepsy

“…GAD65 knockout mice showed increased seizure susceptibility and the genetic deletion of GAD67 caused severe developmental defects and early deaths in mice [ 70 , 73 , 74 , 75 , 76 ]. On the other hand, elevated GAD67 expression in the hippocampal CA3 region by using recombinant adeno-associated virus (AAV) significantly decreased the seizure generation in temporal lobe epilepsy models [ 77 ]. These results indicate that the manipulation of GABA level by targeting the GAD enzyme can be a therapeutic strategy for maintaining appropriate excitability of neurons and treating epilepsy [ 77 ].…”

“…On the other hand, elevated GAD67 expression in the hippocampal CA3 region by using recombinant adeno-associated virus (AAV) significantly decreased the seizure generation in temporal lobe epilepsy models [ 77 ]. These results indicate that the manipulation of GABA level by targeting the GAD enzyme can be a therapeutic strategy for maintaining appropriate excitability of neurons and treating epilepsy [ 77 ]. VGAT is also responsible for synaptic inhibition at GABAergic inhibitory synapses and its dysfunction is related to epilepsy.…”