The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.

The locus coeruleus (LC), the main noradrenergic center in the brain, participates in many neural functions, as diverse as memory and motor output, and is severely affected in several neurodegenerative disorders of the CNS. GDNF, a neurotrophic factor initially identified as dopaminotrophic, was found to be expressed in several targets of central noradrenergic neurons in the adult rat brain. Grafting of genetically engineered fibroblasts expressing high levels of GDNF prevented > 80% of the 6-hydroxydopamine-induced degeneration of noradrenergic neurons in the LC in vivo. Moreover, GDNF induced a fasciculated sprouting and increased by 2.5-fold both tyrosine hydroxylase levels and the soma size of lesioned LC neurons. These findings reveal a novel and potent neurotrophic activity of GDNF that may have therapeutic applications in neurodegenerative disorders affecting central noradrenergic neurons, such as Alzheimer's, Parkinson's, and Huntington's diseases.

Neural stem cells (NSCs) have been proposed as tools for treating neurodegeneration because of their capacity to give rise to cell types appropriate to the structure in which they are grafted. In the present work, we explore the ability of NSCs to stably express transgenes and locally deliver soluble molecules with neuroprotective activity, such as glial cell line-derived neurotrophic factor (GDNF). NSCs engineered to release GDNF engrafted well in the host striatum, integrated and gave rise to neurons, astrocytes, and oligodendrocytes, and maintained stable high levels of GDNF expression for at least 4 months. The therapeutic potential of intrastriatal GDNF-NSCs grafts was tested in a mouse 6-hydroxydopamine model of Parkinson's disease. We found that GDNF-NSCs prevented the degeneration of dopaminergic neurons in the substantia nigra and reduced behavioral impairment in these animals. Thus, our results demonstrate that NSCs efficiently express therapeutic levels of GDNF in vivo, suggesting a use for NSCs engineered to release neuroprotective molecules in the treatment of neurodegenerative disorders, including Parkinson's disease.

Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF-, NT-3-, or NT-4/5-secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF-secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67-, preproenkephalin-, and preprotachykinin A-but not prodynorphinexpressing neurons were protected by grafting of NT-3-or NT-4/5-secreting cells but with less efficiency than the BDNF-secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease. Key Words: Rat striatum-Quinolinate-Grafting-Survival. J. Neurochem. 75, 2190 -2199 (2000).Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene (MacDonald and Gusella, 1996;Mangiarini et al., 1996). Its predominant pathological feature is a massive and progressive degeneration of striatal output neurons without substantial loss of striatal interneurons and afferents (for review, see DiFiglia, 1990). Intrastriatal injection of quinolinate (QUIN), an NMDA receptor agonist, replicates many neurochemical, histological, and behavioral features of HD (Beal et al., 1986;DiFiglia, 1990). Striatal projection neurons containing enkephalin are affected to a greater extent than substance P-containing neurons, and those neurons surviving the lesion express reduced levels of their mRNAs both in HD (Reiner et al., 1988;Richfield et al., 1995) and after QUIN injections (Pérez-Navarro et al., 1999a,b). Excitotoxicity and apoptosis have been suggested to be involved in the degeneration of neurons in HD (Thomas et al., 1995;Petersén et al., 1999) and after QUIN injection (Ferrer et al., 1995;Portera-Cailliau et al., 1995;Hughes et al., 1996). Furthermore, intrastriatal QUIN injections have been found to induce huntingtin mRNA (Carlock et al., 1995) and protein (Tatter et al., 1995), providing a possible link between the QUIN model and HD.Members of the neurotr...

Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6-hydroxydopamine-induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival-promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson's disease.