GDNF applied to the MPTP-lesioned nigrostriatal system requires TGF-β for its neuroprotective action
Introduction
Glial cell line-derived neurotrophic factor (GDNF) was originally discovered as a potent neurotrophic factor with prominent survival-promoting and restorative effects for midbrain dopaminergic neurons in vitro and in animal models of Parkinson’s disease (PD; Lin et al., 1993, Tomac et al., 1995a, Unsicker et al., 1999, Airaksinen and Saarma, 2001, Krieglstein, 2004). This discovery, together with promising results obtained in primate models of PD (Gash et al., 1996), raised great expectations to develop GDNF into a potent drug to cure or ameliorate the pathological symptoms of PD. However, despite the positive outcome of small open-label clinical trials (Gill et al., 2003, Patel et al., 2005, Slevin et al., 2005), other studies including a recent double-blind placebo-controlled study failed to demonstrate efficacy and safety of GDNF (Kordower et al., 1999, Nutt et al., 2003, Lang et al., 2006, Sherer et al., 2006).
We have previously shown for several neuron populations that GDNF does not have neurotrophic activity on its own, but requires TGF-β to act as a neurotrophic factor (Krieglstein et al., 1998, Schober et al., 1999). On a molecular level, we have provided evidence that TGF-β is required to target the GDNF co-receptor α1 (GFRα1) to the cell surface (Peterziel et al., 2002). In addition, GDNF and TGF-β signaling seem to converge both at the PI3-K/Akt as well as ERK pathways. Cooperativity of GDNF and TGF-β is also important at the in vivo level.
Preganglionic sympathetic neurons in the spinal cord die following destruction of a major target, the adrenal medulla, but survive when supplemented with GDNF. We found that neutralization of endogenous TGF-β abolished this protective effect of GDNF (Schober et al., 1999). Together, these data suggest the hypothesis that GDNF may require TGF-β for mediating its neurotrophic effect also in the lesioned nigrostriatal system, possibly including human PD. Consistent with this notion, chromaffin cells, which co-store and co-release GDNF and TGF-β (Krieglstein et al., 1998), have been shown to exert restorative effects on the lesioned nigrostriatal system in animal models and human PD (Bohn et al., 1987, Goetz et al., 1991, Luquin et al., 1999, Espejo et al., 2001, Toledo-Aral et al., 2003).
We now report that neutralizing antibodies against TGF-βs applied together with GDNF to the MPTP-lesioned striatum abolish the neuroprotective effect of GDNF. Specifically, the GDNF-mediated protection of striatal dopamine (DA) levels, relative density of striatal tyrosine hydroxylase (TH)-ir fibers, and the number of TH-ir nigral neurons are significantly reduced or abolished by neutralizing endogenous TGF-β. TGF-β antibodies are not toxic and do not interfere with internalization and retrograde transport of iodinated GDNF from the striatum to the substantia nigra (SN). MPTP treatment causes a 3-fold increase in striatal TGF-β2 mRNA, consistent with the notion that members of the TGF-β family may be available and effective in this system. In summary, we conclude that trophic effects of GDNF in the MPTP-lesioned nigrostriatal DA system require endogenous TGF-β.
Section snippets
Animals
A total of 133 male adult (body weight: 20–25 g; 4–6 months old) C57Bl6 mice, 16 male adult (4–6 months old) Engrailed-1 heterozygous (En-1 +/−) mice (129J/C57Bl6), and 16 corresponding wildtype littermates were used for dopamine (DA) measurements, RT-PCR, quantitative histological analysis of striatum and substantia nigra (SN), and for retrograde transport studies (see Table 1).
MPTP administration
Mice received three intraperitoneal injections at 24 h intervals of MPTP
TGF-β antibodies abolish protection of DA levels by GDNF in the MPTP lesioned striatum
It has previously been shown that GDNF exerts both protective and reparative actions in the MPTP-lesioned nigrostriatal DA system (Tomac et al., 1995a, Gash et al., 1996, Cheng et al., 1998). We have reported that the neurotrophic and protective effects of GDNF are crucially dependent on the simultaneous presence of TGF-β, both in neuronal cell cultures and in an in vivo lesion model of the peripheral nervous system (Krieglstein et al., 1998, Schober et al., 1999). To begin to explore whether
Discussion
The present study provides evidence that TGF-β acts as an essential co-factor of GDNF in a classic model of PD, the MPTP-lesioned mouse (Dauer and Przedborski, 2003, Schober, 2004). The evidence is based on the neutralization of endogenous TGF-β using a monoclonal antibody that recognizes all three TGF-β isoforms and is highly specific for TGF-β (Krieglstein et al., 2000).
Acknowledgments
We thank Richard Hertel, Jutta Fey, Martina Scharpff and Angelika Brüntgens for valuable technical assistance, and Daniel Gherbassi for providing engrailed heterozygous mice (En-1 +/−). Supported by the German Research Foundation (through DFG-Forschungszentrum CMPB (to KK); DFG SFB 636 TP A5 (to KU); NIH grant EY 12841 (to CSvB).
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