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.

Changes in BDNF expression after different types of brain insults are related to neuroprotection, stimulation of sprouting, and synaptic reorganization. In the cerebral cortex, an autocrine-paracrine mechanism for BDNF has been proposed because the distribution patterns of BDNF and TrkB expression are almost identical. Moreover, cortical BDNF is anterogradely transported to the striatum, suggesting a role of BDNF in the functional interaction between the two brain regions. Here we have examined the expression of this neurotrophin in the cerebral cortex after various striatal lesions. Intrastriatal injection of quinolinate, kainate, 3-nitropropionic acid, or colchicine increased BDNF mRNA levels in cerebral cortex. In contrast, stimulation of neuronal activity in the striatum did not change cortical BDNF expression. Both excitatory amino acids increased BDNF expression in neurons of cortical layers II/III, V, and VI that project to the striatum. Moreover, grafting a BDNFsecreting cell line prevented both the loss of striatal neurons and the cortical upregulation of BDNF induced by excitotoxins. Because retrograde transport in the corticostriatal pathway was intact after striatal lesions, our results suggest that striatal damage upregulates endogenous BDNF in corticostriatal neurons by a transneuronal mechanism, which may constitute a protective mechanism for striatal and/or cortical cells.

The use of stem cells for reconstructive or neuroprotective strategies can benefit from new advances in neuroimaging techniques to track grafted cells. In the present work, we analyze the potential of a neural stem cell (NSC) line, which stably expresses the glial cell line-derived neurotrophic factor (GDNF) and the firefly luciferase gene (GDNF/Luc-NSC), for cell therapy in a Huntington's disease mouse model. Our results show that detection of light photons is an effective method to quantify the proliferation rate and to characterize the migration pathways of transplanted NSCs. Intravenous administration of luciferine, the luciferase substract, into the grafted animals allowed the detection of implanted cells in real time by an optical neuroimaging methodology, overpassing the limits of serial histological analyses. We observed that transplanted GDNF/Luc-NSCs survive after grafting and expand more when transplanted in quinolinatelesioned nude mouse striata than when transplanted in nonlesioned mice. We also demonstrate that GDNF/Luc-NSCs prevent the degeneration of striatal neurons in the excitotoxic mouse model of Huntington's disease and reduce the amphetamine-induced rotational behavior in mice bearing unilateral lesions.

Supranigral infusions of the TrkB-receptor-preferring neurotrophins BDNF or NT-4/5 augment locomotor behaviours, pars compacta firing rates and striatal dopamine metabolism. However these actions of BDNF or NT-4/5 may involve other neurotransmitter systems in addition to dopamine neurons in the substantia nigra. We thus investigated the effects of 2-week supranigral infusions of BDNF or NT-4/5 on rat peptidergic striatonigral neurons and nigral GABAergic neurons. Radioimmunoassay revealed that BDNF and NT-4/5 elevated substantia nigra levels of substance P (by 46 and 57% respectively) and substance K (by 64 and 81%). In addition, BDNF elevated substance K by 59% in a nigral projection area, the superior colliculus. NT-4/5 elevated dynorphin A in the substantia nigra (by 52%) and met-enkephalin in substantia nigra and globus pallidus (by 89%). None of these neuropeptides were altered in the striatum. Consistent with these findings, supranigral infusions of BDNF elevated the mRNA for preprotachykinin A in striatal neurons. In the same animals, glutamic acid decarboxylase (GAD)67 mRNA was increased by 48% in the substantia nigra. The cross-sectional area of GAD67-positive neuronal somata in the BDNF-infused nigra was increased by 59%, and 70% of nigral GABAergic neurons had a cross-sectional area > 550 microns2, whereas 95% of the neurons in vehicle-infused animals had cross-sectional areas < 550 microns2. Thus, supranigral infusions of BDNF or NT-4/5 increase tachykinin mRNA and protein levels within striatonigral neurons and increase the size and GAD67 mRNA expression levels of nigral GABAergic neurons. These results suggest that BDNF or NT-4/5 may modify the output of the basal ganglia not only through effects on dopamine neurons but also by increasing neurotransmission in striatonigral peptidergic and nigral GABAergic pathways.