Exploring the Potential Protective Role of FGF-20 in the Rodent Substantia Nigra Pars Compacta

We previously showed that treatment with fibroblast growth factor 20 (FGF-20) was neuroprotective in a rat model of Parkinson’s disease (PD) bearing a full 6-hydroxydopamine (6-OHDA)-induced lesion (1). Although genetic associations between variants of the FGF-20 gene and PD (2) suggest endogenous FGF-20 itself has a role in enhancing survival of dopaminergic neurones in the substantia nigra pars compacta (SNpc), such an action has not yet been demonstrated in vivo. The aim of this study was to explore the effects of modulation of FGF-20 on neuronal survival in vivo using a partial 6-OHDA lesion model of PD. In addition, we examined which FGF receptors (FGFR1, 3 or 4) resided on dopaminergic neurones and identified which cell types could provide a source of endogenous FGF-20 within the SNpc.

Under general anaesthesia, adult male Sprague Dawley rats received unilateral intra-nigral injections of 6-OHDA (4µg) to lesion the nigrostratal tract (2). In the first study, rats received supranigral infusion 1 day prior to and 5 days post 6-OHDA lesion with FGF-20 (1µg/day or 2.5 µg/day (n=6)) or vehicle (aCSF containing 100 ng/ml rat serum albumin; n=9) (1). In the second study, rats were treated with the broad spectrum FGFR antagonist, PD17307 (2 mg/kg s.c.; n=6) or vehicle (10% DMSO, 10% PEG200 in PBS, pH 7.6; n=7) for 3 days prior to and 5 days post lesion. Brains were removed on day 12 (FGF-20 study) or day 7 (FGFR antagonist study) and immunohistochemistry performed to quantify the number of remaining tyrosine hydroxylase (TH) positive neurones in the SNpc and terminal density in the striatum (1). TH-positive cell counts (SNpc) and optical density measures (striatum) were averaged for the intact and lesioned hemispheres from a minimum of 3 sections per animal. Levels in the lesioned hemisphere were then expressed as % of intact hemisphere. Data are given as mean ± SEM (n animals) and were analysed using unpaired t-tests. Fluorescent immunohistochemistry was performed on 8 µm sections of SNpc from paraffin-embedded brains of naive adult male Sprague Dawley rats to examine co-localisation of FGFRs or FGF-20 with TH, GFAP (astrocytic marker) or Iba-1 (microglial marker).

Supranigral infusion with the higher dose of FGF-20 (2.5 µg/day for 6 days) provided modest, but significant protection against the 6-OHDA-induced lesion. Striatal TH and the number of TH-positive neurones were reduced 54% and 71%, respectively in saline-treated animals but only 33% and 50%, respectively in the 2.5 µg/day FGF-20 group. In contrast, treatment with the FGFR antagonist, PD17307, to block any actions of endogenous FGF-20, increased the 6-OHDA lesion. In the SNpc, for example, 6-OHDA produced 76±6% (n=7) TH-positive cell loss in vehicle-treated rats versus 92±2% (n=6) loss in PD17307-treated rats (P=0.04). Immunohistochemical studies revealed the presence of FGFR1, 3 and 4 in TH-positive neurones, although FGFR3 was restricted to nuclei. FGF-20 was present in the SNpc where it co-localised with GFAP, but was absent from TH-positive neurones and Iba1-positive microglia.

In summary, these data show that increasing FGF-20 levels can protect against partial 6-OHDA induced lesion of the nigrostriatal tract, an action likely mediated through activation of FGFR1 or FGFR4 found on TH-positive neurones, but not FGFR2 (exclusive to astrocytes (3)) or FGFR3 (nuclear only). Antagonism of FGFRs exacerbated the 6-OHDA lesion, supporting a protective role for endogenous FGF-20 in the SNpc. Given FGF-20 was only co-localised with GFAP in the SNpc, we propose that FGF-20 may enhance neuronal survival in a paracrine manner via release from neighbouring astrocytes.

1) Sleeman I et al. (2012). Neuropharmacology 63:1268-1277.

2) Zhu R et al. (2014). Neurol Sci 35:1889-1894.

3) Chadashvili T et al. (2006). J Comp Neurol 498:1-15