Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo

Abstract

Neuropeptide Y (NPY) is a 36-amino-acid peptide that attenuates seizure activity following direct infusion or adeno-associated virus (AAV)-mediated expression in the central nervous system. However, NPY activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2 receptor, thought to mediate the antiseizure activity. Therefore, we investigated if recombinant adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that express and constitutively secrete full-length NPY (AAV-FIB-NPY) or NPY13-36 (AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizures. One week later, all animals received kainic acid (10 mg kg(-1), intraperitoneally), and the latencies to wet dog shakes and limbic seizure behaviors were determined. Although both control and vector-treated rats developed wet dog shake behaviors with similar latencies, the latencies to class III and class IV limbic seizures were significantly prolonged in both NPY- and NPY13-36-treated groups. Thus, AAV-mediated expression and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic seizures, and provides a platform for delivering therapeutic peptide fragments with increased receptor selectivity.

Figure 1

The effects of AAV-FIB-NPY and AAV-FIB-NPY13-36 vectors on the expression of limbic seizure behaviors. Rats received bilateral infusions of AAV-FIB-NPY (2 µl per 20 min; 3.3 × 10¹² viral particles per ml; N = 7) or AAV-FIB-NPY13-36 (2 µl per 20 min; 3.0 × 10¹² viral particles per ml; N = 7) into the piriform cortex as described previously. Seven days later both vector-treated groups and an untreated control group (N = 8) received an intraperitoneally injection of kainic acid (10 mg kg⁻¹). The latencies to limbic seizure behaviors were determined for 240 min post-kainic acid. Seizures were scored according to the Racine Motor Seizure Grading Scale. Briefly, class I seizures were scored when rats exhibited facial twitches and chewing, class II when head nodding, class III when contralateral forelimb clonus was observed and class IV when bilateral forelimb clonus and rearing was observed. All groups developed wet dog shakes with the same latencies, indicating the uniformity of kainic acid administration across the different groups. In marked contrast, however, onset of class III and class IV seizures was significantly delayed or completely blocked in rats receiving either AAV-FIB-NPY or AAV-FIB-NPY13-36 compared to control (*t-test; P < 0.01). In fact, only 3/7 AAV-FIB-NPY-treated rats and 1/7 AAV-FIB-NPY13-36-treated rats developed any class III or class IV seizure behaviors over the entire 240 min observation period. All eight rats in the untreated control group developed class III and class IV seizures within 90 min. AAV, adeno-associated virus; FIB, fibronectin; NPY, neuropeptide Y.

Figure 2

The in vivo presence of FIB-NPY and FIB-NPY13-36 mRNA 1 week after vector infusion into the piriform cortex. The appropriate 208 bp FIB-NPY (lane A) or 172 bp FIB-NPY13-36 (lane C) product is present in the injected piriform cortex, while no product was found in an area slightly distal to the piriform cortex (lane B). Omission of the reverse transcriptase step (lanes D and E) indicated the absence of contaminating viral DNA. The left outside lane contains a 100 bp DNA stepladder (Invitrogen, Carlsbad, CA, USA) with the size indicated on the left. Rats received bilateral infusions of AAV-FIB-NPY (2 µl per 20 min; 3.3 × 10¹² viral particles per ml; N = 2) or AAV-FIB-NPY13-36 (2 µl per 20 min; 3.0 × 10¹² viral particles per ml; N = 2) into the piriform cortex as described previously. Then, 1 week later, the animals received an overdose of pentobarbital (100 mg kg⁻¹, intraperitoneally) and were subsequently decapitated. The brain was removed, and the piriform cortex was dissected out. The tissue was stored in RNAlater (Ambion, Austin, TX, USA) at −20°C. Subsequently, the RNA was extracted from the tissue (Promega SV-40 total RNA Isolation kit; Promega, Madison, WI, USA) and reverse transcribed using AMV reverse transcriptase and oligo (dT) primers. The subsequent PCR primers were designed to span the FIB-NPY (and thus the FIB-NPY13-36) sequence, which can only be derived from the rAAV vector (FIB, 5′-CTAGCAGTCCTGTGCCTG; NPY and NPY13-36, 3′-GCTCAATATCTCTGTCTGGTG). AAV, adeno-associated virus; FIB, fibronectin; NPY, neuropeptide Y; rAAV, recombinant adeno-associated virus.