Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption

Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.