CAMP pretreatment increases the number of dopamine neurons in intrastriatal grafts

Abstract

Neuroprotective signalling pathways are under investigation in order to improve the survival of transplanted embryonic dopamine (DA) neurons as a potential therapy for Parkinson's disease. Since cyclic AMP (cAMP)‐induced activation of cAMP‐response element binding protein (CREB) is associated with neuroprotection, we investigated whether cAMP could be used to enhance DA neuron survival and phenotype in a grafting paradigm. We previously reported that dibutyryl (db) cAMP increased the number of tyrosine hydroxylase positive (TH+) neurons in mesencephalic cultures. However, addition of dbcAMP to cell suspensions immediately prior to grafting did not increase the number of DA neurons in intrastriatal grafts. To increase the duration of cAMP exposure, we adapted a rotary, reaggregate culture system which allows protracted culture of embryonic mesencephalic cells without loss of viability. Cell suspensions were prepared and allowed to reaggregate for 24 h. Cultures were then incubated ± dbcAMP for an additional 72 h. The spheres were harvested and transplanted bilaterally into the intact striatum of male Fischer 344 rats. Grafts from dbcAMP‐pretreated spheres contained three times more TH+ cells than grafts from spheres not exposed to dbcAMP. However, due to a high degree of variance, this increase did not reach statistical significance (p = 0.10). Protein harvested from an aliquot of these spheres was subjected to Western blot analysis which revealed that treatment of reaggregate spheres with dbcAMP for 72 h increased TH expression by 78% and increased Bcl2 expression by 47%. Mechanisms underlying these results are currently under investigation.