We have shown previously that low concentrations of noradrenaline (NA) confer long-term but partial protection to tyrosine hydroxylase (TH ϩ ) dopaminergic neurons by reducing spontaneously occurring oxidative stress. We demonstrate here that the effect of NA is strongly enhanced by cAMPelevating agents, in particular forskolin (FK), through a mechanism that does not involve activation of adrenoceptors. FK also enhanced the neuroprotective action of antioxidants that mimic the trophic effects of NA, such as trolox and pyrocatechol, but was totally ineffective by itself, suggesting that inhibition of oxidative stress was a required step to reveal the cAMP-dependent mechanism. Neuroprotection afforded by FK was rapidly reversible, optimal when the treatment was initiated in the early phase of the culture and exquisitely specific to dopaminergic neurons. FK stimulated the phosphorylation of extracellular signal-activated kinases (ERK) 1/2 in a subpopulation of dopaminergic neurons, suggesting that the mitogen-activated protein kinase (MAPK) pathway was involved in the effects of cAMP-elevating agents. Accordingly, inhibition of the upstream kinases of ERK 1/2 by 2Ј-amino-3Ј-methoxyflavone (PD98059) not only suppressed MAPK activation caused by FK but also abolished the survival promoting activity that this compound exerts on TH ϩ neurons. PD98059 did not reduce, however, the trophic effects provided by NA alone. Surprisingly, the archetypal cAMP-dependent protein kinase was apparently not responsible for ERK 1/2 activation. The data suggest that the MAPK signaling pathway plays a key role in the trophic effects that cAMP elevating agents and NA cooperatively exert on TH ϩ neurons.

Cyclic AMP-elevating agents are highly effective in preventing the loss of dopaminergic neurons that occurs spontaneously in neuronal-glial mesencephalic cultures. We demonstrate here that cAMP causes a concomitant decline in the number of dividing non-neuronal cells, suggesting that inhibition of proliferation contributes to neuroprotection. Consistent with this hypothesis, a transient treatment with the antimitotic cytosine arabinoside, at concentrations that induce long-term repression of glial cell proliferation, mimicked the neuroprotective action of cAMP and also obviated the need for the cyclic nucleotide. Treatment with cAMP-elevating agents reduced the population of OX-42-positive microglial cells and the number of immature astrocytes expressing vimentin and low levels of the astrocytic marker glial fibrillary acidic protein. The effect on the immature astrocytes, however, seemed essential for neuroprotection. Ciliary neurotrophic factor and leukemia inhibitory factor, which stimulate astrocyte differentiation without reducing cell proliferation, failed to reproduce the protective effects of the cyclic nucleotide. Cyclic AMP did not operate by counteracting the action of the astrocyte mitogen epidermal growth factor or by reducing activation of the mitogen-activated protein kinase signaling pathway. The neuroprotective and antiproliferative actions of cAMP, however, were closely mimicked by olomoucine and roscovitine, potent inhibitors of the cyclindependent kinase CDK1 that are structurally related to cAMP. Measurement of CDK1 activity confirmed that neuroprotection was closely correlated with inhibition of this kinase by cAMP. In summary, neuroprotection of mesencephalic dopaminergic neurons by cAMP most probably requires the repression of presumptive astrocytes through inhibition of CDK1.

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Levodopa, the major treatment for patients with Parkinson's disease, has been shown to induce a variety of compensatory effects, including facilitation of sprouting by dopaminergic neurons, in experimental animals with lesions leading to denervation of the striatum. To better understand the cellular and molecular environment where most of these compensatory changes take place, in particular elements that might contribute to the recovery of dopaminergic innervation, we have constructed a differential expression library enriched in transcripts from the striata of rats with lesions of the medial forebrain bundle treated with levodopa for 6 months. We have used this library to screen an expression array of rat genes representing the major cell functions, and have identified several that are involved in neurotrophic mechanisms and plasticity. We have confirmed the differential expression of selected transcripts by non-radioactive in situ hybridization, and report that the growth factor pleiotrophin, myelin basic protein and calmodulin are overexpressed in the denervated striatum of levodopa-treated rats.

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