Abstracts

Rationale: Long-lasting neuropeptide Y (NPY) overexpression in rat hippocampus is induced by local application of adeno-associated viral vector (rAAV) with chimeric 1/2 serotype (rAAV1/2-NPY) under control of the neuron-specific enolase (NSE) promoter. rAAV1/2-NSE-NPY injected rats showed reduced seizures and delayed kindling. We optimized rAAV vector acting either at the capsid or promoter level to increase the efficiency of cell transduction and enhance NPY-mediated anticonvulsant activity without interfering with physiological functions.Methods: Cassettes carrying the human NPY gene under the control of cytomegalovirus enhancer/chicken-beta-actin (CBA) promoter flanked by AAV2 ITRs were cross-packaged into capsids of AAV type 1 or 1/2 (a mixture of AAV1 and AAV2 capsid proteins at a 1:1 ratio). The new vectors were compared with the rAAV1/2-NSE-NPY vector. Rats were injected with rAAV-NPY vectors (3µl 5.2x1012) into the septal and temporal aspects of the hippocampus bilaterally or with the corresponding rAAV-Empty-cassette vectors or saline (controls). Four weeks after rAAV delivery, behavioral testing of learning and memory (two platforms discrimination water maze task and passive avoidance test) and anxiety (exploration of a white-black open-field) were carried out. After one week, the same rats were intrahippocampally injected with 40 ng of kainic acid and the onset, number of seizures and time spent in seizures were evaluated by EEG analysis. Neuroprotection and inflammation were evaluated by immunohistochemistry.Results: No differences were observed in the behavioral tests between experimental groups and their controls except for rats injected with chimeric rAAV1/2-CBA-NPY vector. These rats showed a significant delay in learning compared to their respective controls in the two platforms discrimination test. rAAV1/2-NSE-NPY or rAAV1/2-CBA-NPY rats showed on average a reduction of 40-50% in number of seizures and of 65% in the time spent in seizures compared to controls. rAAV1-CBA-NPY rats showed a larger reduction (by 80%) in the number and time spent in EEG seizures. In all vector–injected rats, anticonvulsant activity was associated with increased NPY immunostaining in hilar interneurons and mossy fibers, however rAAV1/2-CBA-NPY rats showed additional upregulation of NPY in CA1 pyramidal neurons. Additional NPY staining in the inner and outer molecular layers of the dentate gyrus and in stratum oriens CA3 was observed in rAAV1-CBA-NPY rats. Neuronal injury in the CA3 area and hilus was reduced in all experimental groups overexpressing NPY. Vector injection per se did not induce cell toxicity or inflammation.Conclusions: Long-lasting NPY overexpression mediated by rAAV1-CBA-NPY vector in the hippocampus provides stronger anticonvulsant activity than rAAV1/2 vectors, neuroprotection and it does not alter behavioral outcomes related to physiological functions.