Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo

Abstract

Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

Fig. 1.

Electrophoretic analysis of recombinant human GDF-15/MIC-1 protein in Sf9 cells. A, Immunoblotting of cell lysate from infected (1) and noninfected (2) insect cells with purified GDF-15/MIC-1 antiserum. The gel was run under reducing conditions. B,Coomassie blue staining of purified recombinant GDF-15/MIC-1 (1) (reducing conditions). C,Immunoblotting of purified recombinant GDF-15/MIC-1 homodimers under nonreducing conditions with purified GDF-15/MIC-1 antiserum (1). MW, Low molecular weight markers.

Fig. 2.

Localization of GDF-15/MIC-1 in the CNS.A, Immunoblotting of human CSF with purified GDF-15/MIC-1 antiserum. B, Dark-field image showsin situ hybridization of adult rat choroid plexus with a rat-specific GDF-15/MIC-1 antisense RNA probe. C, RT-PCR of different rat (P0) brain regions (pons, medulla oblongata, cortex, hippocampus, striatum), dorsal root ganglia (drg), cultured primary astrocytes (astr.), the oligodendroglial cell line OLI-neu (OLI), and purified oligodendroglial progenitor cells (O-2A).D, Immunoblotting of rat brain tissues (P0) and cells with purified GDF-15/MIC-1 antiserum. Locations of molecular weight marker bands are provided on the left side of each figure.

Fig. 3.

Photomicrographs of cell cultures established from the E14 rat midbrain floor at DIV 7. Panels show staining with monoclonal antibodies against TH (A, C,E) or GFAP (B, D, F), respectively. Cultures were run as controls (A, B), or treated with GDNF (10 ng/ml; C, D), or GDF-15/MIC-1 (1 ng/ml; E,F). Scale bar, 50 μm.

Fig. 4.

In vitro neurotrophic effects of GDF-15/MIC-1. GDF-15/MIC-1 was assayed on rat midbrain DAergic neurons (A, B), rat serotonergic raphe neurons (C), and chick DRG neurons (D). A, Numbers of mesencephalic TH-positive neurons at DIV 7. C, No factors;BC, baculovirus control, i.e., noninfected cells;1, 2, 3, cultures treated with 0.01, 0.1, and 1 ng/ml GDF-15/MIC-1, respectively;G, GDNF (10 ng/ml). B, Numbers of mesencephalic TH-positive neurons at DIV 8, after intoxication with 100 μm Fe²⁺. C, No factors; NT-4 (10 ng/ml); GDF-15/MIC-1 (1 ng/ml). C, Numbers of TpOH and 5,7 DHT-positive cells at DIV 4 in cultures established from rat E14 raphe. C, Control; 1,2, Cultures treated with 5 (1) or 10 ng/ml (2) GDF-15/MIC-1. D, Numbers of chick (E8) DRG neurons at DIV 2. C, Control, 1, 2, 3, cultures treated with 1, 5, or 10 ng/ml GDF-15/MIC-1; G, cultures treated with GDNF (10 ng/ml); N, cultures treated with NGF (10 ng/ml). Data are given as means ± SEM (n = 3). All experiments were performed in triplicate and repeated at least three times. p values derived from Student's ttest are *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5.

Photomicrographs of cryosections through the left SNpc. Sections were processed for TH immunocytochemistry. Animals were treated as follows: A, vehicle only; B, 6-OHDA only (10 d); C, 6-OHDA plus 10 μg of GDF-15/MIC-1 (10 d); D, 6-OHDA plus 40 μg of GDF-15/MIC-1 (10 d); E, 6-OHDA only (1 month);F, 6-OHDA plus 40 μg of GDF-15/MIC-1 (1 month). Scale bar, 200 μm.