Regulation of natural cell death in dopaminergic neurons of the substantia nigra by striatal glial cell line-derived neurotrophic factor in vivo.

Dopamine (DA) neurons of the substantia nigra undergo a developmental cell death event that is biphasic, with peaks just after birth and at postnatal day 14. As envisioned by neurotrophic theory, this cell death is likely to be regulated by target interactions because it is augmented by their disruption. However, the nature of the trophic molecules mediating this regulation are unknown. We showed in vitro that glial cell line-derived neurotrophic factor (GDNF) is able to suppress apoptotic death in DA neurons in postnatal primary culture. We now demonstrate in vivo that administration of GDNF into the striatal target is able to suppress apoptosis. Consistent with a possible physiologic role for endogenous striatal GDNF in regulating this event, two anti-GDNF neutralizing antibodies augment cell death. These antibodies augment cell death only during the first (immediately postnatal) phase of the biphasic death event. We conclude that GDNF is the leading candidate for a target-derived neurotrophic factor for the regulation of the early phase of natural cell death in DA neurons.

Regulation of the development of mesencephalic dopaminergic systems by the selective expression of glial cell line-derived neurotrophic factor in their targets.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore dopamine (DA) neurons in injury models and is being evaluated for the treatment of Parkinson's disease. Nevertheless, little is known of its physiological role. We have shown that GDNF suppresses apoptosis in DA neurons of the substantia nigra (SN) postnatally both in vitro and during their first phase of natural cell death in vivo. Furthermore, intrastriatal injection of neutralizing antibodies augments cell death, suggesting that endogenous GDNF plays a role as a target-derived factor. Such a role would predict that overexpression of GDNF in striatum would increase the surviving number of SN DA neurons. To test this hypothesis, we used the tetracycline-dependent transcription activator (tTA)/tTA-responsive promoter system to create mice that overexpress GDNF selectively in the striatum, cortex, and hippocampus. These mice demonstrate an increased number of SN DA neurons after the first phase of natural cell death. However, this increase does not persist into adulthood. As adults, these mice also do not have increased dopaminergic innervation of the striatum. They do, however, demonstrate increased numbers of ventral tegmental area (VTA) neurons and increased innervation of the cortex. This morphologic phenotype is associated with an increased locomotor response to amphetamine. We conclude that striatal GDNF is necessary and sufficient to regulate the number of SN DA neurons surviving the first phase of natural cell death, but it is not sufficient to increase their final adult number. GDNF in VTA targets, however, is sufficient to regulate the adult number of DA neurons.

Anatomical basis of glial cell line-derived neurotrophic factor expression in the striatum and related basal ganglia during postnatal development of the rat.

There is increasing evidence that glial cell line-derived neurotrophic factor (GDNF) plays a role as a limiting, striatal target-derived neurotrophic factor for dopamine neurons of the substantia nigra pars compacta (SNpc) by regulating the magnitude of the first phase of postnatal natural cell death which occurs in these neurons. While it has been shown that GDNF mRNA is relatively abundant in postnatal striatum, the cellular basis of its expression has been unknown. We therefore used nonradioactive in situ hybridization and immunohistochemistry to examine the cellular basis of GDNF mRNA and protein expression, respectively, in postnatal striatum and related structures. We found that GDNF mRNA is expressed within medium-sized striatal neurons. Expression in glia was not observed. At the protein level, regionally, GDNF expression in striatum was observed in striosomal patches, as previously described. At a cellular level a few neurons were observed, but they do not account for the striosomal pattern. This pattern is predominantly due to GDNF-positive neuropil. Some of this neuropil arises from tyrosine hydroxylase-positive nigro-striatal dopaminergic afferents. Astrocytic processes do not appear to contribute to the striosomal pattern. GDNF-positive fibers are identified not only within intrinsic striatal neuropil, but also in fibers within the major striatal efferent targets: the globus pallidus, the entopeduncular nucleus, and the SN pars reticulata. We conclude that during normal postnatal development, medium-sized neurons are the principal source of GDNF within the striatum.

Glial cell line-derived neurotrophic factor receptor GFRalpha1 is expressed in the rat striatum during postnatal development.

Dopamine neurons of the substantia nigra (SN) undergo a natural cell death event which is biphasic, with peaks at postnatal days (PNDs) 2 and 14. There is growing evidence that GDNF functions as a striatal target-derived neurotrophic factor to regulate the first phase. It has been unknown whether the GDNF receptor, GFRalpha1, may play a role in regulating either phase. To evaluate a possible role for GFRalpha1 we have examined its expression throughout postnatal development in the SN and particularly in the striatum, where its expression has been uncertain. GFRalpha1 mRNA is highly expressed in SN, as previously shown, with highest levels at PND14-28. We find that it is also expressed in striatum with a similar time course, but with a more discrete period of maximal expression between PND10 and PND14. The cellular basis of this maximum of expression is an increased number of GFRalpha1 mRNA-positive medium-sized neurons evenly distributed within the striatum. Immunostaining reveals GFRalpha1 protein-positive neurons with a similar morphology and distribution. We conclude that GFRalpha1 is expressed in striatum maximally late in postnatal development. In this location it may act in trans to influence the viability and development of nigral dopamine neurons.

Histochemical methods for the detection of apoptosis in the nervous system.

Neuroscientists often need to detect neuron death at the light microscope level in tissue sections derived from animal models of neurological disease. In many instances there is a need to detect apoptosis, the most common morphology of programmed cell death. This unit provides two protocols for the detection of apoptosis by immunostaining for either activated forms of caspases or their cleavage products. When used in conjunction with nuclear dyes, these protocols permit visualization not only of caspase activation, but also the nuclear chromatin clumps characteristic of apoptosis. The first protocol utilizes peroxidase-mediated chromogen deposition to visualize antibodies by brightfield microscopy. The second protocol utilizes fluorophores to visualize antibodies by epifluorescence. Double immunofluorescence labeling can be performed to identify the phenotype of cells in which caspases are activated. Not all cell death is apoptotic. Therefore, a third protocol is presented for suppressed silver staining, a useful method to screen for all morphologic forms of cell death.

CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism.

There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.

Ectopic expression of cell cycle markers in models of induced programmed cell death in dopamine neurons of the rat substantia nigra pars compacta.

There is increasing evidence that proteins normally involved in the cell cycle can regulate neuronal programmed cell death (PCD). However, it remains unknown whether cell cycle markers are expressed in normal, postmitotic, postmigratory neurons undergoing PCD in vivo. We have previously shown that natural cell death occurs postnatally in dopamine neurons of the substantia nigra pars compacta (SNpc). PCD can be induced postnatally in these neurons either by intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA) or by medial forebrain bundle (MFB) axotomy. At the time of induction of death in these models, these neurons are long postmitotic and postmigratory. We have studied three cell cycle markers in these models: 5-bromo-2'-deoxyuridine (BrdU) incorporation (a marker of S phase), cdc2 protein expression (a marker of G2 phase), and expression of MPM2 (a marker of M phase), an epitope phosphorylated by cdc2. We report here that postmitotic dopaminergic neurons undergoing PCD in the SNpc following 6-OHDA and axotomy lesions incorporate BrdU and overexpress cdc2, but do not express MPM2. This is the first in vivo evidence that postmitotic dopamine neurons of the SNpc undergoing apoptosis express markers for S phase and G2 phase. These results raise the possibility that cell cycle regulatory proteins may play a role in the demise of dopaminergic neurons in Parkinson's disease, in which PCD has been postulated to play a role.

CEP11004, a novel inhibitor of the mixed lineage kinases, suppresses apoptotic death in dopamine neurons of the substantia nigra induced by 6-hydroxydopamine.

There is much evidence that the kinase cascade which leads to the phosphorylation of c-jun plays an important signaling role in the mediation of programmed cell death. We have previously shown that c-jun is phosphorylated in a model of induced apoptotic death in dopamine neurons of the substantia nigra in vivo. To determine the generality and functional significance of this response, we have examined c-jun phosphorylation and the effect on cell death of a novel mixed lineage kinase inhibitor, CEP11004, in the 6-hydroxydopamine model of induced apoptotic death in dopamine neurons. We found that expression of total c-jun and Ser73-phosphorylated c-jun is increased in this model and both colocalize with apoptotic morphology. CEP11004 suppresses apoptotic death to levels of 44 and 58% of control values at doses of 1.0 and 3.0 mg/kg, respectively. It also suppresses, to approximately equal levels, the number of profiles positive for the activated form of capase 9. CEP11004 markedly suppresses striatal dopaminergic fiber loss in these models, to only 22% of control levels. We conclude that c-jun phosphorylation is a general feature of apoptosis in living dopamine neurons and that the mixed lineage kinases play a functional role as up-stream mediators of cell death in these neurons.