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

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.

An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.

A defective herpes simplex virus type one (HSV-1) vector that contains a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac-7kb) was examined for its capability to target catecholaminergic cell type-specific expression in the CNS. Cell type-specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV-1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac-7kb supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac-7kb supported a 10-fold targeting of beta-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac-7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long-term expression from pTHlac-7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased approximately 30-fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV-1 vector system, the tyrosine hydroxylase promoter enhanced cell type-specific expression and contributed to stable, long-term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.

Expression of the calcium-binding protein, parvalbumin, in cultured cortical neurons using a HSV-1 vector system enhances NMDA neurotoxicity.

Calcium-binding proteins (CaBPs) are a family of proteins having a unique distribution in the brain and are thought to be important in buffering intracellular calcium. Glutamate neurotoxicity is a process by which the over-activation of glutamate receptors can cause the influx of excessive extracellular calcium and neuronal cell death. It has been proposed that neurons containing CaBP may be more resistant to glutamate neurotoxicity due to their increased ability to buffer calcium. Using a herpes simplex virus-1 (HSV-1) vector system we packaged the CaBP gene, parvalbumin, or the marker gene, beta-galactosidase (beta-gal), correctly in viron particles, which were found upon infection to express mRNA specific to these vectors. PC12 and neocortical cultures showed strong immunohistochemical staining for either beta-gal or parv. The cortical cultures stained positively for endogenous glutamate decarboxylase, a marker for GABAergic neurons, but not for endogenous parvalbumin, indicating that parvalbumin was being expressed ectopically from the HSV-1 vector. Interestingly, the expression of parvalbumin increased cortical culture's susceptibility to N-methyl-D-aspartate-induced neurotoxicity. This increase in neurotoxicity was not due to the wild-type virus or the helper virus which accompanies the packaging of these vectors. We speculate that the ectopic expression of parvalbumin in cortical cultures may be increasing glutamate release which in turn increases cell death.