Jukka Hartikka

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

Brain-derived neurotrophic factor (BDNF) and NGF are both expressed by neurons in the hippocampus. In previous studies, it has been demonstrated that both BDNF and NGF mRNA levels are regulated by neuronal activity. Upregulation is predominantly regulated by the glutamate (NMDA and non-NMDA receptors); downregulation, predominantly by the GABA system (Zafra et al., 1990, 1991). In neuronal cultures of the rat hippocampus, potassium depolarization and kainic acid-mediated increases in BDNF and NGF mRNA were eliminated in a dose-dependent manner by the calcium channel blocker nifedipine. Conversely, calcium ionophores (Bay-K8644 and ionomycin) augmented BDNF and NGF mRNA levels by a calmodulin-mediated mechanism. In view of the fact that many potential modulators (conventional transmitters and neuropeptides) of neuronal and astrocytic BDNF and NGF mRNA synthesis may act via the adenylate cyclase system, we studied the effect of forskolin, an activator of adenylate cyclase. Indeed, forskolin enhanced the effects of calcium ionophores and kainic acid on BDNF and NGF mRNA levels. Cytokines, such as interleukin-1 and transforming growth factor-beta 1, which have previously been shown to increase NGF mRNA markedly in astrocytes, were without effect on neuronal BDNF and NGF mRNA levels. In contrast to neuronal cultures, where the regulation of BDNF and NGF mRNA was generally very similar, the regulation in astrocytes was distinctly different. All the cytokines that produce a marked increase in NGF mRNA were without effect on astrocyte BDNF mRNA levels, which under basic conditions were below the detection limit. However, norepinephrine produced a marked elevation of BDNF mRNA in astrocytes, an effect that was further enhanced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)