Activation of CNTF/CNTFRα Signaling Pathway by hRheb(S16H) Transduction of Dopaminergic Neurons In Vivo

Ciliary neurotrophic factor (CNTF) is one of representative neurotrophic factors for the survival of dopaminergic neurons. Its effects are primarily mediated via CNTF receptor α (CNTFRα). It is still unclear whether the levels of CNTFRα change in the substantia nigra of Parkinson’s disease (PD) patients, but CNTF expression shows the remarkable decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc), suggesting that the support of CNTF/CNTFRα signaling pathway may be a useful neuroprotective strategy for the nigrostriatal dopaminergic projection in the adult brain. Here, we report that transduction of rat SNpc dopaminergic neurons by adeno-associated virus with a gene encoding human ras homolog enriched in brain (hRheb), with an S16H mutation [hRheb(S16H)], significantly upregulated the levels of both CNTF and CNTFRα in dopaminergic neurons. Moreover, the hRheb(S16H)-activated CNTF/CNTFRα signaling pathway was protective against 1-methyl-4-phenylpyridinium-induced neurotoxicity in the nigrostriatal dopaminergic projections. These results suggest that activation of CNTF/CNTFRα signaling pathway by specific gene delivery such as hRheb(S16H) may have therapeutic potential in the treatment of PD.


Introduction
Ciliary neurotrophic factor (CNTF), a neurotrophic cytokine belonging to the interleukin-6 family, is expressed in diverse brain regions [1]. CNTF regulates several functions, such as gene expression, cell survival, and differentiation in a variety of neuronal cells [2,3]. These are mediated by tripartite receptor complexes, such as leukemia inhibitory factor receptor β, glycoprotein 130, and CNTF-specific ligand-binding receptor α-subunit (CNTFRα) [4]. Expression of CNTF and CNTFRα can increase in the brain following injury [5][6][7]; however, the levels of CNTF are reduced in dopaminergic neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients [8] and treatment with recombinant human CNTF preventes the degeneration of dopaminergic neurons [9]. Moreover, the loss of neurotrophic factors such as glial cell linederived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) with CNTF may be involved in the pathogenesis of brain diseases [8,10,11]. These observations suggest that the support of neurotrophic factors, such as CNTF, GDNF, and BDNF, may be useful to protect neurons against neurodegenerative diseases, and their induction can be potential therapeutic targets for PD.
In a previous study, we have shown that transduction of human ras homolog enriched in brain (hRheb) with a mutation of serine to histidine at position 16 [hRheb(S16H)] exerted neurotrophic effects in dopaminergic neurons via the activation of mammalian target of rapamycin complex 1 (mTORC1), resulting in the protection and restoration of the nigrostriatal dopaminergic projection in a neurotoxin model of PD [12,13]. Furthermore, we have recently reported that hRheb(S16H) expression could induce the production of BDNF and GDNF, which is neuroprotective in dopaminergic neurons in a neurotoxin model of PD [14]. These results suggest that activation of the signaling pathways involved in cell survival by a specific gene delivery, such as hRheb(S16H), may be a useful strategy for protecting the nigrostriatal dopaminergic system in the adult brain.
It is still unclear whether hRheb(S16H) transduction of dopaminergic neurons can induce the production of additional survival factors such as CNTF and CNTFRα, which are also involved in protection of the nigrostriatal dopaminergic projection in the adult brain. Therefore, in the present study, we examined whether hRheb(S16H) as an activator for production of CNTF and CNTFRα and if this contributed to neuroprotection against 1-methyl-4phenylpyridinium (MPP + )-induced neurotoxicity on the nigrostriatal dopaminergic projection in the adult rat brain.

Institutional review of animal protocols
Forty Sprague Dawley rats (10 weeks old, 220-240 g) were obtained from Daehan Biolink (Eumseong, Korea). Rats were housed in a controlled environment (temperature, 22 ± 1°C; humidity, 50 ± 1%) and fully provided with food and water. All surgical experiments were performed in accordance with approved animal protocols and guidelines established by the Animal Care Committee of Kyungpook National University (No. KNU 2012-37). All surgeries were performed under anesthesia by intraperitoneal injection of chloral hydrate (360 mg/kg; Sigma, St. Louis, MO), and all efforts were made to minimize suffering and reduce the number of animals used.

Production of adeno-associated virus (AAV) viral vectors
All vectors used in these studies were AAV1 serotype. AAVs with a gene encoding hRheb (S16H) or green fluorescent protein (GFP), used as a control, were produced by the University of North Carolina Vector Core as previously described [12][13][14]. The genomic titer of hRheb (S16H) and GFP were 2 × 10 12 and 1 × 10 12 viral genomes/ml, respectively.

Intranigral AAV injection
Rats received a unilateral injection of AAV-GFP or AAV-hRheb(S16H) into the right SN as previously described [14]. Coordinates were calculated according to Paxinos and Watson [15] (AP: -6.0 mm; ML: -2.3 mm; DV: -7.6 mm, relative to the bregma). After the injection, the needle was left in place for an additional 5 min before being slowly retracted. The viral vector suspension (2.0 μl) was injected at a rate of 0.1 μl/min over 20 min.

Stereological estimation
As previously described [16][17][18][19], the total number of TH-positive neurons was counted in the various animal groups using the optical fractionator method performed on an bright-field microscope (Olympus Optical, BX51, Tokyo, Japan) using Stereo Investigator software (MBF Bioscience, Williston, VT). Dopaminergic neurons in the SN were quantitatively expressed as a percentage of the number of TH-positive neurons in the ipsilateral side compared to the contralateral control side.

Quantitative determination of striatal TH immunoperoxidase staining
The densitometric analysis in the rat striatum (STR) was examined as previously described [12,14,20]. TH-positive fiber innervation in the STR was quantitatively expressed as a percentage of the optical density on the ipsilateral side compared with the contralateral control side.

Western blot analysis
Brain tissues were prepared for western blotting as previously described [14,20]. Briefly, rat SN tissues were homogenized and centrifuged at 4°C for 20 min at 14,000 g. The supernatant was transferred to a fresh tube and the concentration was determined using a BCA kit (Bio-Rad Laboratories, Hercules, CA). Proteins were separated using gel electrophoresis and transferred on to polyvinylidene difluoride membranes (Millipore) using an electrophoretic transfer system (Bio-Rad Laboratories). Membranes were incubated overnight at 4°C with specific primary antibodies: anti-β-actin (1:4000; Cell Signaling, Beverly, MA), anti-CNTF (1:1000; R&D systems) and anti-CNTFRα (1:10000; R&D systems). After washing, membranes were incubated with secondary antibodies (Amersham Biosciences, Piscataway, NJ) and the blots were developed with ECL western blotting detection reagents (Amersham Biosciences). For semiquantitative analyses, the density of the immunoblot bands was measured with the Computer Imaging Device and accompanying software (Fuji Film, Tokyo, Japan).

Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). Multiple comparisons among the groups were performed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc analysis. All statistical analyses were performed using the Sigma Stat software (Systat Software, San Leandro, CA).

Increases in the levels of CNTF and CNTFRα by hRheb(S16H) transduction of dopaminergic neurons in vivo
To examine whether hRheb(S16H) expression induced an increase in the levels of CNTF and CNTFRα in the SN, brain sections were immunostained with anti-CNTF or anti-CNTFRα 4 weeks after the intranigral injection of AAV-GFP or AAV-hRheb(S16H). Immunoperoxidase staining showed that the increases in CNTF and CNTFRα were observed in the SNpc following hRheb(S16H) transduction of dopaminergic neurons, compared with the contralateral control side and GFP-treated rats ( Fig. 2A). The increased CNTF and CNTFRα were localized within dopaminergic neurons in the SN (Fig. 2B). Consistent with the immunostaining results, western blot analysis showed that hRheb(S16H) transduction of dopaminergic neurons significantly increased both CNTF and CNTFRα expression in the SN, compared with the control groups ( Fig. 2C; p < 0.01).
hRheb(S16H)-induced CNTF and CNTFRα contribute to the protection of the nigrostriatal dopaminergic projection in a neurotoxin model of PD We next examined whether hRheb(S16H)-induced CNTF and CNTFRα were neuroprotective in a neurotoxin model of PD. Rats received a unilateral injection of neutralizing antibodies against CNTF into the SN 3 weeks after AAV-hRheb(S16H) treatment and then received an injection of MPP + into the MFB. Brain tissues treated with either neutralizing antibodies or MPP + alone were used as a control for neurotoxicity. Immunostaining for TH, which was confirmed 1 week after the intranigral injection, showed that rats treated with CNTF neutralizing antibodies alone had no change in the number of TH-positive neurons or optical density of striatal TH immunostaining (Fig. 3A). However, the protective effects of hRheb(S16H) against  MPP + -induced neurotoxicity were inhibited by its treatment (Fig. 3A). When quantified and expressed as a percentage of TH-positive neurons in the ipsilateral SN compared to the contralateral control, there were 58% and 88% TH-positive neurons in rats injected with MPP + alone and MPP + in the presence of hRheb(S16H), respectively ( Fig. 3B; p < 0.001 and p = 0.750, respectively, vs. contralateral controls). This indicated that hRheb(S16H) was neuroprotective in the MPP + -treated animal model of PD (Fig. 3B; p < 0.001 vs. MPP + alone). The administration of neutralizing antibodies for CNTF significantly attenuated the hRheb(S16H)-induced neuroprotection in the MPP + -treated model of PD [ Fig. 3B; p < 0.001 vs. contralateral controls, p = 0.010 vs. hRheb(S16H) + MPP + ]. As previously described [12,14], hRheb(S16H) transduction of dopaminergic neurons significantly increased TH immunoreactivity in the STR, compared with contralateral controls (Figs. 3A and C; p < 0.05 vs. contralateral controls). Similar to the results in the SN, there were 30% and 86% TH-positive fibers in rats injected with MPP + alone and MPP + in the presence of hRheb(S16H), respectively ( Fig. 3C; p < 0.001 and p = 0.946, respectively, vs. contralateral controls). This further indicated hRheb(S16H)-induced neuroprotection in the MPP + -treated model of PD (Fig. 3C; p < 0.001 vs. MPP + alone). However, CNTF neutralization significantly inhibited hRheb(S16H)-induced neuroprotection [ Fig. 3C; p < 0.001 vs. contralateral controls, p = 0.001 vs. hRheb(S16H) + MPP + ].
To assess the role of CNTFRα in hRheb(S16H)-induced neuroprotection, we further analyzed additional rats that received an intranigral injection of neutralizing antibodies against CNTFRα (Fig. 4A). Similar to CNTF neutralization, this treatment significantly attenuated hRheb(S16H)-induced neuroprotection in both dopaminergic neurons and fibers in the MPP + -treated model of PD [Figs. 4B and C; p = 0.016 and p < 0.001 for TH-positive neurons and fibers, respectively, vs. hRheb(S16H) + MPP + ]. However, anti-CNTFRα alone did not significant effect neurotoxicity (Figs. 4B and C; p = 0.946 and p = 0.299 for TH-positive neurons and fibers, respectively, vs. contralateral controls). Therefore, these results indicate that the activation of CNTF/CNTFRα signaling pathway by hRheb(S16H) transduction of dopaminergic neurons may contribute to the neuroprotection on the nigrostriatal dopaminergic projection in the adult brain.

Discussion
Rheb is a small GTPase belonging to the Ras superfamily of guanine nucleotide-binding proteins. It is a key upstream regulator of mTORC1 activity, which is involved in multiple cellular An increase in CNTF contributes to the hRheb(S16H)-induced neuroprotection of the nigrostriatal dopaminergic projection. (A) Rats received an intranigral injection of neutralizing antibodies for CNTF 3 weeks after AAV-hRheb(S16H) injection, and then they received an intra-MFB injection of MPP + 10 min after treatment with neutralizing antibodies. Representative coronal sections of TH immunoperoxidase staining showed that hRheb(S16H) transduction of dopaminergic neurons induced neurotrophic effects as previously described [12,14], protected the nigrostriatal dopaminergic projection from MPP + -induced neurotoxicity. However, treatment with neutralizing antibodies for CNTF (CNTF-NA) attenuated the neuroprotective effects of hRheb(S16H)induced neuroprotection. Neutralizing antibodies alone were not neurotoxic in normal brains. CON, contralateral control side; EXP, ipsilateral injected side. Scale bars, 500 μm and 10 μm in the SN, respectively, and 500 μm in the STR. (B and C) The quantitative analysis of TH-positive neurons and fibers. Consistent with the immunostaining results, CNTF neutralization significantly inhibited hRheb(S16H)-induced neuroprotection in the MPP + -treated model of PD. All values are expressed as a percentage of the results in the ipsilateral SN and STR, respectively, compared with the contralateral controls (One-way ANOVA and Tukey post-hoc analysis; n = 4, each group).
doi:10.1371/journal.pone.0121803.g003 functions, including protein synthesis, cell growth, proliferation, survival, and synaptic plasticity [21][22][23][24]. Our previous studies have shown that a constitutively active form of Rheb, such as hRheb(S16H), could induce neurotrophic effects in SNpc dopaminergic neurons, stimulate the production of dopamine in the nigrostriatal dopaminergic system, and induce neuroprotective and restorative effects via the activation of the mTORC1 signaling pathway in animal models of PD [12,13]. In addition, the present study shows an additional mechanism of hRheb (S16H)-induced neuroprotection in a neurotoxin model of PD, suggesting that hRheb(S16H) transduction of dopaminergic neurons may have therapeutic potential as a treatment for PD.
CNTF immunoreactivity is attenuated in dopaminergic neurons in the SN of PD patients [8]. Moreover, levels of CNTF and CNTFRα could increase in the brain following injury, such as focal cerebral ischemia and intracerebral hemorrhage [5][6][7]. These results suggest that the activation of survival signaling pathway by the induction of neurotrophic factors through an auto-/paracrine system after brain damage may be compensatory responses against neurodegeneration, which could be involved in PD [11], although it cannot fully protect the neurons in damaged brain regions. Furthermore, while neutralizing antibodies for CNTF and CNTFRα were not neurotoxic in this study, these data support the hypothesis that functional suppression of CNTF/CNTFRα signaling pathway may be involved in the neurodegeneration of CNTFRα neutralization inhibits the protective effects of the nigrostriatal dopaminergic projection by hRheb(S16H) transduction against MPP + -induced neurotoxicity. (A) Neutralizing antibodies for CNTFRα (CNTFRα-NA) were injected in rats 3 weeks after the intranigral injection of AAV-hRheb(S16H). Following this, rats were injected into the MFB with MPP + . Representative coronal sections of the SN and STR of TH immunoperoxidase staining showed that CNTFRα neutralization attenuated the protective effects induced by hRheb(S16H) against MPP + neurotoxicity. CON, contralateral control side; EXP, ipsilateral injected side. Scale bars represent 500 μm and 10 μm in the SN, respectively, and 500 μm in the STR. (B and C) The quantitative analysis of TH-positive neurons and fibers demonstrated that treatment with neutralizing antibodies for CNTFRα significantly decreased the hRheb(S16H)-induced neuroprotection on the nigrostriatal projection. All values are expressed as a percentage of the results in the ipsilateral SN and STR, respectively, compared with the contralateral controls (One-way ANOVA and Tukey post-hoc analysis; n = 4, each group).
doi:10.1371/journal.pone.0121803.g004 dopaminergic neurons following neurotoxin treatment in the brain. Therefore, the enough supplies of neurotrophic factors such as CNTF could be crucial for the protection and maintenance of the nigrostriatal dopaminergic projection in the adult brain. However, similar to GDNF and BDNF [14], systemic delivery of CNTF as a therapeutic agent is blocked by the blood-brain barrier and intracerebral injection is not therapeutically valid due to its short halflife [25]. Nevertheless it has been shown to be neuroprotective against the degeneration of dopaminergic neurons [8,10,11]. These results suggest that the development of novel strategies for production or delivery of CNTF into the target region may be useful for prevention and therapy in PD. The results in the present study showed that viral vector-mediated hRheb (S16H) transduction increased the expression of CNTF and CNTFRα in dopaminergic neurons in the SN of rat brains (Fig. 2). Furthermore, treatment with specific neutralizing antibodies for CNTF and CNTFRα attenuated the protective effects of hRheb(S16H) against MPP +induced neurotoxicity in the adult brain (Figs. 3 and 4). These results suggest that the activation of CNTF/CNTFRα signaling pathway contributes to hRheb(S16H)-induced neuroprotection; however, we cannot exclude the possibility that hRheb(S16H)-induced CNTF may be Schematic representation of hRheb(S16H)/CNTF and CNTFRα signaling pathways. The serine at position 16 of hRheb is sensitive to tuberous sclerosis complex (TSC) GTPase activation. hRheb(S16H), which has a mutation of the serine to histidine, is resistant to this activation and consequently cause its persistent GTP-bound, activated state [12]. The accumulation of hRheb(S16H) stimulates the expression of CNTF and its receptor in dopaminergic neurons, which can consequentially protect the nigrostriatal dopaminergic projection from neurotoxicity, even though it is obscure whether CNTF produced by hRheb (S16H) is involved in other potential mechanisms for protection of dopaminergic neurons (dotted arrow). involved in other potential mechanisms, such as the induction of other neurotrophic factors, against neurotoxicity in dopaminergic neurons (Fig. 5).
In conclusion, in addition to the important role of hRheb(S16H) as an activator for the production of GDNF and BDNF in adult dopaminergic neurons [14], the present results show that specific gene delivery hRheb(S16H) can activate CNTF/CNTFRα signaling pathway in dopaminergic neurons in the SN and protect against MPP + -induced neurotoxicity. This may provide a useful therapeutic strategy for the neuroprotection of the nigrostriatal dopaminergic projection in the adult brain.