Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease

Abstract

Neurotrophic factors are promising candidates for the treatment of Parkinson's disease (PD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a novel evolutionarily conserved family of neurotrophic factors. We examined whether MANF has neuroprotective and neurorestorative effect in an experimental model of PD in rats. We also studied the distribution and transportation of intrastriatally injected MANF in the brain and compared it with glial cell line-derived neurotrophic factor (GDNF). Unilateral lesion of nigrostriatal dopaminergic system was induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Amphetamine-induced turning behavior was monitored up to 12 weeks after the unilateral lesion. The local diffusion at the injection site and transportation profiles of intrastriatally injected MANF and GDNF were studied by immunohistochemical detection of the unlabeled growth factors as well as by autoradiographic and gamma counting detection of (125)I-labeled trophic factors. Intrastriatally injected MANF protected nigrostriatal dopaminergic nerves from 6-OHDA-induced degeneration as evaluated by counting tyrosine hydroxylase (TH)-positive cell bodies in the substantia nigra (SN) and TH-positive fibers in the striatum. More importantly, MANF also restored the function of the nigrostriatal dopaminergic system when administered either 6 h before or 4 weeks after 6-OHDA administration in the striatum. MANF was distributed throughout the striatum more readily than GDNF. The mechanism of MANF action differs from that of GDNF because intrastriatally injected (125)I-MANF was transported to the frontal cortex, whereas (125)I-GDNF was transported to the SN. Our results suggest that MANF is readily distributed throughout the striatum and has significant therapeutic potential for the treatment of PD.

Figure 1.

a–c, MANF protects against 6-OHDA induced toxicity in rats in vivo. a, Purified MANF. SDS-PAGE gel stained with Coomassie brilliant blue is shown. Lane 1, Molecular weight markers; lane 2, 5 μg of purified MANF; lane 3; 10 μg of purified MANF. b, c, Effect of intrastriatal injection of vehicle, MANF (3, 10, or 30 μg), or GDNF (10 μg) on amphetamine-induced rotation. Rats received vehicle, MANF, or GDNF in the striatum 6 h before a lesion was induced by intrastriatal 6-OHDA (8 μg). Amphetamine-induced behavior was measured for 2 h at 2 and 4 weeks after lesion. b, Cumulative amphetamine-induced rotations. c, Amphetamine-induced rotations expressed as ipsilateral rotations per 5 min in different treatment groups at 4 weeks after lesion. b, c, Means ± SEM are shown; n = 7–13 in each group. Tukey–Kramer's post hoc analysis after one-way ANOVA, *p < 0.05, **p < 0.01 versus the corresponding control.

Figure 2.

MANF protects TH-positive cell bodies in the substantia nigra and TH-immunoreactive fibers in the striatum from 6-OHDA. a, TH-positive cell bodies in the SN analyzed at 4 weeks after lesion. In the vehicle-treated rat, intrastriatal 6-OHDA caused loss of ∼34% of the TH-positive cell bodies in the SN compared with the intact side. The protective effect of MANF was significant at the dose of 10 μg. b–e, Photomicrographs of sections from the midbrain showing TH-immunohistochemical staining of the SN. b–e, TH-positive cells in the SNpc of an intact rat (b) or of a 6-OHDA-lesioned rat pretreated with either vehicle (c), GDNF (10 μg) (d), or MANF (10 μg) (e). f, Density of TH-positive fibers in the striatum. Optical density of striatal sections was measured at three rostrocaudal levels. Optical density was reduced by 36% (compared with the intact side) in the vehicle-treated rats. MANF protected TH-positive fibers in the striatum. The GDNF-treated group did not differ from the vehicle group. g, h, Photomicrographs of striatal TH-positive fibers in an intact rat (g) or pretreated either with vehicle (h), GDNF (10 μg) (i) or MANF (10 μg) (j). Scale bar: b–e, 270 μm. a, f, Means ± SEM are shown; n = 8–13 in each group. Tukey–Kramer's post hoc analysis after one-way ANOVA, **p < 0.01 versus the corresponding control.

Figure 3.

Relationships between behavioral and morphological measures. a–c, Correlation between amphetamine-induced rotation and TH immunoreactivity in brain, assessed by simple linear regression analysis. For each experimental group, the mean number of ipsilateral turns of each rat at 2 weeks after lesion is plotted against the mean number of ipsilateral turns at 4 weeks after lesion (a). The mean number of ipsilateral turns at 4 weeks after lesion was plotted against the mean number of TH-positive cells in the SN (expressed as percentage of the contralateral intact side) (b) or against the mean striatal TH-positive fiber density (expressed as the mean of the intact side) (c).

Figure 4.

Neurorestorative effects of MANF. Rats were administered 6-OHDA (20 μg) unilaterally in the striatum. Four weeks later, the rats received a single injection of either vehicle, GDNF (10 μg), or MANF (10 μg). The rotational behavior was measured 1 week before and 2, 4, 6, and 8 weeks after the injection of the growth factors. a, Cumulative amphetamine-induced ipsilateral rotations. Means ± SEM are show; n = 6–10 in each group. b, Combined cumulative amphetamine-induced ipsilateral rotations measured at 3, 6, 8, 10, and 12 weeks after lesion. c, Number of TH-positive cells in the SNpc. Means ± SEM are shown; n = 6–9 in each group. Tukey–Kramer's post hoc analysis after one-way ANOVA, ***p < 0.001, **p < 0.01 versus the corresponding control.

Figure 5.

Distribution of MANF and GDNF after a single striatal injection. Rats were administered either MANF (10 μg) or GDNF (10 μg) unilaterally in the striatum. Representative immunohistochemical staining for MANF (a) and GNDF (b) in a coronal section through the brain 24 h after an injection of the neurotrophic factor. c, The diffusion profile of MANF also differs from the one of GDNF with statistical significance (the arrow in c points out the site for protein injection in relation to bregma). Means ± SEM are shown; n = 6 in both groups. Statistical significance in c was estimated with repeated-measures one-way ANOVA followed by Tukey–Kramer's post hoc test. **p < 0.01.

Figure 6.

Distribution of ¹²⁵I-GDNF and ¹²⁵I-MANF after intrastriatal injection. a, Photomicrograph of the autoradiographic film. Film shows differences in transportation between MANF and GDNF. After intrastriatal injections of ¹²⁵I-MANF and ¹²⁵I-GDNF, a strong labeling was seen in the injection site (striatum) in sections processed from both two trophic factor-injected brains. ¹²⁵I-MANF is transported to the cortical brain area, but not to the SN. After ¹²⁵I-GDNF injection, strong signal was detected in the SN. Radioactive signal was detected also in the cortex after injection of ¹²⁵I-GDNF. Brain slice figures modified from the rat brain atlas of Paxinos and Watson (1997). A/P 2.70 corresponds to cortex (olfactory bulb), A/P 1.00 corresponds to striatum (injection site), A/P −0.8 corresponds to dorsal striatum, A/P −3.30 corresponds to hippocampus, and A/P −5.80 corresponds to substantia nigra. b, c, Six microliters of ¹²⁵I-labeled neurotrophic factor was injected into the left striatum with PBS or (in a competitive manner) with at least 100-fold excess of either unlabeled MANF or GDNF. Rats were perfused 24 h later and brain samples were immediately performed for gamma counting. b, Radioactivity in the substantia nigra. c, Radioactivity in frontal cortical area. The results are expressed as mean ± SEM; n = 8 in MANF group, n = 10 in GDNF group.