Modulation of rat rotational behavior by direct gene transfer of constitutively active protein kinase C into nigrostriatal neurons

Abstract

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKCbetaII (PkcDelta) were developed. PkcDelta exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, PkcDelta caused a long-lasting, activation-dependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkcDelta in a small percentage of nigrostriatal neurons (approximately 0.1-2%) was sufficient to produce a long-term (>/=1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained PkcDelta, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphine-induced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D2-like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphine-induced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.

Fig. 1.

Western blots and immunocytochemical analyses of PkcΔ, PkcΔGG, and PkcΔAA in S. cerevisiae and PC12 cells. A, Replicate sets of aliquots of SDS-solubilizedS. cerevisiae and PC12 cells were subjected to SDS-polyacrylamide gel electrophoresis, followed by blot immunolabeling analysis. Protein staining is shown in the left panel(PONCEAU), and immunoreactivities with the indicated antibodies are shown in the right panels.Lane 1, Purified rat brain PKC; lanes 2–5, extracts from S. cerevisiae harboring no YEp51 plasmid, YEp51pkcΔ, YEp51pkcΔAA, or YEp51pkcΔGG, respectively; lanes 6–8, extracts from PC12 cells 1 d after infection with pHSVlac, pHSVpkcΔ, or pHSVpkcΔGG, respectively. The molecular weight markers are β-gal, β-galactosidase (116 kDa); phos, phosphorylase b (97 kDa); BSA, bovine serum albumin (67 kDa);cat, catalase (60 kDa); GDH, glutamate dehydrogenase (55 kDa); oval, ovalbumin (42 kDa); andCA, carbonic anhydrase (29 kDa). B, C,Flag-IR positive cells 1 d after PC12 cells were infected with either pHSVpkcΔ (B) or pHSVpkcΔGG (C). Scale bars, 333 μm.

Fig. 2.

Apomorphine-induced rotational behavior after microinjection of pTHpkcΔ, control vectors, or PBS. The number of rotations was measured during each 5 min period for 1 hr after apomorphine administration. The rats were tested for apomorphine-induced rotational behavior before gene transfer (pretest). From 4 to 7 d later, pTHpkcΔ, pTHpkcΔGG, pHSVpkcΔ, pTHlac, or PBS was injected into the midbrain. Apomorphine-induced rotational behavior was measured at 1, 2, and 3 weeks after gene transfer. On week 4, fluphenazine, a DA receptor antagonist, was administered 3 hr before apomorphine-induced rotational behavior was tested (Bruno et al., 1985). On week 5, rats in the pTHpkcΔ and pTHlac groups were retested for apomorphine-induced rotational behavior. A, For the rats in the pTHpkcΔ group, shown is the average number of rotations performed during each 5 min period in each of five tests (pretest andweeks 1–4). These rotations were in the ipsilateral direction. B, For the rats in the pTHpkcΔ and control groups, shown is the average over three tests (weeks 1–3) of the average number of rotations performed during each 5 min period.

Fig. 3.

TH-IR positive cells and flag-IR positive cells with neuronal morphology at either 4 d or 6 weeks after gene transfer with pTHpkcΔ. pTHpkcΔ was injected into the midbrain; either 4 d (A–C) or 6 weeks (D–H) later (after the behavioral analysis), adjacent sections were analyzed for TH-IR and flag-IR positive cells. The SNc was located in sections by visualizing the TH-IR positive cells (A). A low-power view of an adjacent section (B) shows flag-IR positive cell bodies and proximal processes in SNc, but not in SNr. Three flag-IR positive cell bodies are indicated by arrows, and a high-power view (C) shows that these three cells display neuronal morphology, including neuronal-like processes. D, A low-power view shows ∼12 flag-IR positive cells in the SNc at 6 weeks after gene transfer. Eacharrow indicates one to several flag-IR positive cells, and the flag-IR positive cell indicated by the empty arrow is shown under medium power in E.F, High-power view of a flag-IR positive SNc cell with a large cell body, characteristic of a SNc neuron, from a second rat.G, H, Low- and high-power views of a flag-IR positive SNc cell, with processes characteristic of a SNc neuron, from a third rat. SNc, Substantia nigra pars compacta;SNr, substantia nigra pars reticulata. Scale bars:A and D, 80 μm; B, 45 μm; C, 25 μm; F, 20 μm;G, 50 μm; H, 10 μm.

Fig. 4.

Apomorphine-induced rotational behavior as a function of the number of flag-IR positive SNc cells. The average of the total number of rotations performed during each of three tests (weeks 1–3) is plotted for each of 11 rats that received pTHpkcΔ as a function of the number of flag-IR positive cells in each rat. Least-squares regression analysis of these data (with and without values from the rat indicated by the circled square) yielded correlation coefficients of r = 0.74 (p < 0.01; solid line) andr = 0.89 (p < 0.001;dashed line), respectively.

Fig. 5.

Long-term expression of pkcΔ transcription unit RNA from pTHpkcΔ and pHSVpkcΔ and the persistence of vector DNAs.A, Detection of pkcΔ transcription unit RNA by RT-PCR. At 2 weeks (pTHpkcΔ) or 6 weeks (pHSVpkcΔ) after injection into the midbrain, RNA was isolated from either the midbrain or the cerebellum. The RNA was treated with DNase, and reverse transcription was performed by using a primer from the 3′ untranslated region of the pkcΔ transcription unit. The reverse transcription reaction products were amplified by performing PCR with primers from the pkcΔ transcription unit. The PCR reaction products were detected by Southern blot analysis, using a radiolabeled oligonucleotide from the pkcΔ coding region. The predicted size of the RT-PCR products is 1239 bp.Mb, Midbrain; Cb, cerebellum. B, C, Detection of pkcΔ transcription unit RNA by in situ hybridization. At 2 weeks after the injection of pTHpkcΔ, in situ hybridization was performed with a digoxigenin-labeled probe. Hybridization was visualized by using an alkaline phosphatase-conjugated anti-digoxigenin antibody, and the nuclei were detected by counterstaining with methyl green.B, A camera lucida drawing of a section that was analyzed; the rectangle shows the location of a group of positive cells in the SNc. C, A high-power photomicrograph reveals a number of positive cells. Each of the twoarrows points to an area of hybridization signal proximal to a nucleus. Scale bar, 50 μm. D–G,Detection of vector DNAs by PCR. DNA was extracted from the midbrain in sections adjacent to those used for immunohistochemistry, PCR was performed with primers from the pkcΔ transcription unit, and the PCR reaction products were displayed on an agarose gel. The predicted size of the PCR reaction products is 1003 bp. D, All of the rats were analyzed at 4 d after gene transfer. Lanes 1, 2, pTHpkcΔ or pHSVpkcΔ DNA isolated fromE. coli, respectively; lanes 3–5, three rats that received pHSVpkcΔ; lanes 6–9, four rats that received pTHpkcΔ; lanes 10–12, three rats that received PBS; lane 13, plasmid PKC-II DNA [contains the rat PKCβII cDNA (Knopf et al., 1986)]; lane 14, no DNA; lanes 15, 16, no primers or noTaq polymerase, respectively, with pTHpkcΔ DNA isolated from E. coli. E–G, All of the rats were analyzed at 6 weeks after gene transfer. E, Lane 1, pTHpkcΔ DNA isolated from E. coli;lanes 2, 3, two rats that received pHSVpkcΔ; lanes 4–11, eight rats that received pTHpkcΔ; lane 12, one rat that received PBS; lane 13, plasmid PKC-II DNA; lanes 14, 15, no primers or no Taq polymerase, respectively, with pTHpkcΔ DNA isolated from E. coli. F, Lanes 1,2, pTHpkcΔGG or pTHpkcΔ DNA isolated from E. coli, respectively; lanes 3–6, four rats that received pTHpkcΔ (in addition to the eight rats in E);lanes 7–9, three rats that received pTHpkcΔGG;lane 10, one rat that received PBS. G, ABamHI site is predicted to be present in the PCR reaction products obtained by using pTHpkcΔGG, but not pTHpkcΔ, as the template; digestion with BamHI is expected to result in 288 and 715 bp fragments. The first two nucleotides in theBamHI site represent the last two nucleotides in the Gly codon in pkcΔGG that replaced the evolutionarily conserved Lys codon in pkcΔ. Lanes 1, 2, pTHpkcΔGG DNA isolated from E. coli, either undigested or digested with BamHI, respectively; lanes 3–6, two rats that received pTHpkcΔ, either undigested (lanes 3, 5) or digested with BamHI (lanes 4, 6); lanes 7–10, two rats that received pTHpkcΔGG, either undigested (lanes 7, 9) or digested withBamHI (lanes 8, 10).