Triggering endogenous neuroprotective mechanisms in Parkinsons disease studies with a cellular model

Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA

Summary. Glial cell line-derived neuro-trophic factor (GDNF) has been implicated in the protection of dopamine (DA) neurons from oxidative stress in animal models of Parkinson's disease (PD). We have now shown that GDNF can also protect against the effects of 6-hydroxydopamine (6-OHDA) in a dopaminergic cell line and in cultures of primary DA neurons prepared from rat sub-stantia nigra (SN). This appears to involve a rapid and transient increase in the phospho-rylation of several isoforms of extracellular signal-regulated kinase (ERK). Our evidence indicates that ERK activation also can be modulated by reactive oxygen species (ROS), including those generated by endogenous DA. Identification of the ways by which these pathways can be triggered should provide insights into the pathophysiology of PD, and may offer useful avenues for retarding the progression of the disorder.

Introduction

Although PD is likely to involve the loss of many types of neurons, it is the loss of DA neurons of the SN that appears responsible for the motor symptoms typically used to define the disorder. Thus, reducing the loss of these cells should provide substantial clinical benefit. Evidence suggests that one reason for SN degeneration is an excess of oxidative stress. We have used an experimental model of PD that involves treating adult rats with an intracerebral, unilateral injection of 6-OHDA to produce a high level of such stress. Normally this would result in a marked reduction in the use of the contralateral limbs and the loss of DA neurons in the ipsilateral nigrostriatal projection. However, working with Tim Schallert and his colleagues at the University of Texas, Austin, we found that these consequences could be attenuated by placing the ipsilateral forelimb in a cast for one week immediately before or after the 6-OHDA treatment, thereby forcing the use of the normally affected limb (Tillerson et al., 2001; Cohen et al., 2003). We now wish to understand the processes that underlie these phenomena in the hope that this will lead to a rational basis for reducing disease progression.

GDNF can attenuate the death of dopaminergic cells normally caused by exposure to oxidative stress

Exercise had already been shown by Carl Cotman and his colleagues at the University of California, Irvine, to increase trophic factors in the brain (Cotman and Bechtold, 2002). Thus, we hypothesized that forced motor activity reduced the vulnerability of DA neurons through an increase in the avail ability of such factors. One likely candidate was GDNF. In 1993, GDNF was identified as a survival factor for DA neurons in culture, and this observation was soon followed by the demonstration that the neurotrophic factor also could protect DA neurons in adult animals against the effects of DA toxins such as 6-OHDA (Bohn, 1999). Moreover, Annie Cohen and Amanda Smith in our group observed that forced exercise caused an increase in GDNF in the contralateral striatum that peaks after 3 days of treatment (Cohen et al., 2003). Thus, we have set about to determine how GDNF protects DA neurons from oxidative stress.

We have begun our studies with two in vitro models. Yun Min Ding, Juliann Jaumotte, and Armando Signore observed that we could produce a specific degeneration of dissociated DA neurons from the SN region of rat pups (P0) if the cultures were briefly exposed to 6-OHDA (40-500 mM; 15 min). This effect was attenuated by the addition of GDNF (100ng/ml) to the med ium (Ding et al., 2004). Using the dopaminergic cell line MN9D, Susana Ugarte, Eva Lin, and Ruth Perez found that 6-OHDA (250 mM, 15 min) also produced a concentration- and time-dependent death of these cells, and that this, too, could be attenuated by the addition of GDNF (10ng/ml) (Ugarte et al., 2003).

GDNF failed to block completely the effects of 6-OHDA in either experimental model. However, we reason that an understanding of the mechanism of the protective effects that are seen will provide us with important insights into determinants of DA neuron survival. Thus, using the MN9D model we have begun to dissect out the intra-cellular events responsible for the GDNF-induced protection that occurs. Eva Lin and Jane Cavanaugh observed that exposure of MN9D cells to GDNF (10-100 ng/ml for 15 min) resulted in the phosphorylation of three isoforms of ERK - ERK1/2 and ERK5, as well as an increase in pCREB (Cavanaugh et al., 2004; Lin et al., 2004).

GDNF & other TFs

GDNF & other TFs

Fig. 1. A simplified version of our hypothesis for the actions of 6-OHDA, DA, and GDNF. Pro-survival pathways in green; pro-death pathways in red. Solid, excitation; dotted, inhibition. Components not specified include are the role of BAD, CREB, and nF-kB, and the importance of protein anchors. The inset is provided to indicate that the kinetics of changes in MAP kinases is critical to their impact on survival, with short term increases in kinase activity often being neuroprotective (shown for ERK) and more prolonged increases often being neurotoxic

(shown for JNK)

Fig. 1. A simplified version of our hypothesis for the actions of 6-OHDA, DA, and GDNF. Pro-survival pathways in green; pro-death pathways in red. Solid, excitation; dotted, inhibition. Components not specified include are the role of BAD, CREB, and nF-kB, and the importance of protein anchors. The inset is provided to indicate that the kinetics of changes in MAP kinases is critical to their impact on survival, with short term increases in kinase activity often being neuroprotective (shown for ERK) and more prolonged increases often being neurotoxic

(shown for JNK)

We believe that the apparent activation of ERK participates in the neuroprotective effects of GDNF since the MEK inhibitor U0126 (5-10 mM) eliminated the protective effects of GDNF against 6-OHDA toxicity in the MN9D cell line (Ugarte et al., 2003).

Members of our research group have found parallels to these findings in animal models. Amanda Smith has examined the effect of forced limb use on activation of ERK1/2 within the nigrostriatal pathway. As in the case of exercise-induced changes in the striatum, Dr. Smith observed that forced limb use increased pERK1/2 in the nigrostriatal pathway associated with the overused forelimb (Smith et al., 2004). In the striatum, forced use increased ERK1/2 (4-fold), which was sustained during the 7-day casting period. In the SN, the increase in pERK1/2 was more gradual, with maximum increases in pERK1/2 of 5-fold at day 7 post-cast (Smith et al., 2004). Niklas Lindgren and Rehana Leak have further shown that the direct injection of GDNF into the striatum produces a marked activation of pERK1/2 in SN neurons within 24 hr. Thus, both in vivo and in vitro data are consistent with the hypothesis that increases in the availability of GDNF protect DA neurons from oxidative stress acting in part via an activation of ERK isoforms (see Fig. 1). Of course, there is still much to be done. For example, we have not yet shown that in vivo blockade of GDNF or ERK signaling will block the neuroprotection we observed in our animal model.

ERK activation may also be triggered by oxidative stress

In our studies of the role of ERK in the neu-roprotective effects of GDNF, we also observed that ERK1/2 and 5 was activated by 6-OHDA itself (Lin et al., 2003; Cavanaugh et al., 2004). To examine this phenomenon more carefully, Eva Lin treated MN9D cells with 6-OHDA (500 mM). We observed that pERK1/2 increased by 25-fold at 15min but returned to baseline by 30 min. After removing the 6-OHDA, a second, smaller, but sustained peak arose by 3-6 hr and persisted for several more hours. This late pERK1/2 peak was correlated with activation of cas-pase-3 and cell death. Phosphorylation of ERK's downstream substrate CREB followed the activation profile of ERK1/2.

To begin to determine the relation between ERK phosphorylation and the toxic effects of 6-OHDA, we re-examined the response to 6-OHDA in the presence of U0126 (5 mM). When this inhibitor was added to the medium before and during toxin exposure so as to block the first pERK peak, vulnerability of cells to 6-OHDA was increased 2-fold. No such effect was seen if U0126 was provided after this initial peak (Lin et al., 2003). This suggests that the initial transient activation of ERK after oxidative stress is a compensatory response, functioning to reduce cellular vulnerability.

Jane Cavanaugh has since shown that 6-OHDA increased pERK5 as well as pERK1 /2 and that U0126 inhibits the activation of all three ERK isoforms. Thus, experiments are now in progress to determine which of the isoforms is involved in retarding the toxic effects of oxidative stress in this model.

The role of endogenous DA in neuroprotection

Having observed that 6-OHDA activated ERK isoforms in MN9D cells and that this was associated with a reduction in 6-OHDA-induced toxicity, we reasoned that DA might have a similar impact. It is well known that DA, like 6-OHDA, is highly electroactive and readily oxidizes to form several ROS. Thus, Eva Lin examined the effects of incubating MN9D cells for 24 hr with a-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in DA biosynthesis. We observed that decreasing DA levels in this way was associated with a decrease in pERK1/2. This was associated with an increase in basal cell death and in the toxic effects of 6-OHDA (Lin et al., 2003). Our observations suggest that the vulnerability of DA neurons is regulated in part by changes in DA turnover and that GDNF acts in part through this mechanism.

Can PD be affected via alterations in GDNF and/or activation of ERK signaling?

Clinical trials with GDNF have produced conflicting results. A report appeared in 2003 by Stephen Gill and his colleagues suggesting that GDNF had great promise. This led to a much larger, multi-center blinded study. Unfortunately, that study has been halted because of reports of a lack of clinical improvement and evidence of potentially toxic effects. Yet, given the clear evidence for the efficacy of GDNF in laboratory models as well as some patients, it seems clear that we must pursue an understanding of how this and related molecules exert their neuropro-tective effects. Moreover, studies of trophic factors, oxidative stress, and related intra-cellular signaling cascades may provide insights into how endogenous neuroprotective processes can be stimulated to retard the progression of PD.

Acknowledgements

Thanks to all the members of our research group who contributed to the results presented here. Our work has been supported in part by grants from the National Institutes of Health (AG17476 and NS19608, NS045698, and NS047831), the US Army (DAMD17-03-0479), and the Michael J. Fox Foundation.

References

Bohn MC (1999) A commentary on glial cell line-derived neurotrophic factor (GDNF). From a glial secreted molecule to gene therapy. Biochem Pharmacol 57: 135-142 Cavanaugh JE, Jaumotte JD, Pinchevsky R, Ganabathi R, Lakoski JM, Zigmond MJ (2004) The role of ERK5 in GDNF-mediated neuroprotection of dopaminergic neurons from oxidative stress. Soc Neurosci Abstr Program No. 221. 3 . 2004 SFN Online Abstract Viewer and Hinerary Planner Cohen AD, Tillerson JL, Smith AD, Schallert T, Zigmond MJ (2003) Neuroprotective effects of prior limb use in 6-hydroxydopamine-treated rats: possible role of GDNF. J Neurochem 85: 299-305 Cotman CW, Berchtold NC (2002) Exercise: a behavioral intervention to enhance brain health and plasticity. Trends Neurosci 25: 295-301 Ding YM, Jaumotte JD, Signore AP, Zigmond MJ (2004) Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to do-pamine neurons and the impact of glial cell line-derived neurotrophic factor. J Neurochem 89: 776-787 Lin E, Perez RG, Zigmond MJ (2004) Role of ERK1 /2 activation in dopaminergic cell survival in response to oxidative stress. Soc Neurosci Abstr Program No. 93. 9. 2004 SFN Online Abstract Viewer and Hinerary Planner Smith AD, Slagel SL, Castro SL (2004) Activation of MAP kinases by forced limb use in a 6-hydroxy-dopamine Parkinson's disease rodent model. Soc Neurosci Abstr Program No. 221. 5 . 2004 SFN Online Abstract Viewer and Hinerary Planner Tillerson JL, Cohen AD, Philhower J, Miller GW, Zigmond MJ, Schallert T (2001) Forced limb-use effects on the behavioral and neurochemical effects of 6-hydroxydopamine. J Neurosci 21: 4427-4435 Ugarte SD, Lin E, Klann E, Zigmond MJ, Perez RG (2003) Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase and MEK. J Neurosci Res 73: 105-112

Author's address: M. J. Zigmond, PhD, Department of Neurology, S-526 Biomedical Science Tower, 3500 Terrace St, University of Pittsburgh, Pittsburgh, PA 15213, USA, e-mail: [email protected]

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