Axon guidance molecule expression after cell therapy in a mouse model of Parkinson’s disease

Abstract

Background: Cell therapy is a promising approach for Parkinson’s disease (PD). Others and we have previously shown that transplantation of ventral mesencephalic fetal cells into substantia nigra (SN) in an animal model of PD enables anatomical and functional repair of the degenerated pathway. However, the molecular basis of this repair is still largely unknown.

Objective: In this work, we studied the expression of several axon guidance molecules that may be implicated in the repair of the degenerated nigrostriatal pathway.

Methods: The expression of axon guidance molecules was analyzed using qRT-PCR on five specific regions surrounding the nigrostriatal pathway (ventral mesencephalon (VM), thalamus (Thal), medial forebrain bundle (MFB), nucleus accumbens (NAcc) and caudate putamen (CPu)), one and seven days after lesion and transplantation.

Results: We showed that mRNA expression of specific axon guidance molecules and their receptors is modified in structures surrounding the nigrostriatal pathway, suggesting their involvement in the axon guidance of grafted neurons. Moreover, we highlight a possible new role for semaphorin 7A in this repair.

Conclusion: Overall, our data provide a reliable basis to understand how axons of grafted neurons are able to navigate towards their targets and interact with the molecular environment in the adult brain. This should help to improve the efficiency of cell replacement approaches in PD.

Keywords

Axon guidance molecule expression substantia nigra transplantation adult brain plasticity

1 Introduction

PD is a chronic progressive neurodegenerative disorder characterized mainly by loss of dopaminergic neurons of the SN inducing a degradation of the nigrostriatal pathway. Pathological hallmarks of this disease include a subsequent decrease in the level of dopamine in the target striatal region, the appearance of motor and non-motor symptoms and the accumulation and propagation of Lewy bodies and Lewy neurites in different brain regions (Desplats et al., 2009; Obeso et al., 2010). Patients with PD are treated with L-Dopa, a dopamine precursor, or with dopamine agonists (Worth, 2013). While these pharmacological treatments do alleviate some of the numerous motor symptoms, theireffectiveness decreases dramatically with time. Another therapeutic approach is the deep brain stimulation using a stimulant probe permanently installed within the subthalamic nucleus of PD patients. This electrophysiological procedure using high-frequency stimulation has shown to improve most cardinal motor features of PD, including resting tremor and has benefited greatly to patients (see review by Miocinovic et al., 2013). However, none of these two main approaches restores the physiological loops within the basal ganglia. In this way, cell replacement therapies, aiming at restoring the main degenerated pathway and increasing dopamine levels in the target striatum, have recently received a renewed attention. Indeed, several studies, including ours, showed that homotopic intranigral transplants of dopaminergic VM cells derived from E12.5 GFP-transgenic donors, are able to produce proper anatomical and functional restoration of the nigrostriatal pathway in mice (Gaillard et al., 2009; Thompson et al., 2009). Extensive striatal innervations observed in these models provided evidence of the permissive capacity of the adult brain for axonal growth. Indeed, axons of grafted neuron were able to grow out of the transplant a few days after the intranigral transplantation of VM cells in the SN. Then, axons followed their path in the diencephalon, ventrally to the Thal, and connected to the dorsolateral striatum from the 7th day after transplantation (Gaillard et al., 2009). This suggests a role for guidance cues in driving axonal projections of the transplanted dopaminergic cells along their proper pathway to their targetregion.

During embryonic development, axonal guidance cues are essential to guide dopaminergic axons growing out of the SN pars compacta to connect the dorsolateral striatum. Several members of the four canonical families of axon guidance molecules are involved in the establishment of this mesostriatal pathway such as netrin-1, ephrins, semaphorins, and Slits (see review by Prestoz et al., 2012). For example, in the telencephalon, netrin-1 has been documented to promote axonal growth of VM dopaminergic neurons in vitro (Lin et al., 2005). Recently, netrin-1 was also shown to exert an important role in the topographical patterning of dopaminergic axonal projections originating from the SN and the ventral tegmental area. Indeed, it allows their connection onto respectively dorsomedial and ventrolateral striatal zones, through a differential attraction effect on the two populations (Li et al., 2014). In addition, our group has also reported that a gradient of ephrin-A5 may contribute to the graded distribution of dopaminergic axons in the striatum (Deschamps et al., 2009). Specific patterns of ephrin-A2 and ephrin-A3 expression in this structure similarly conduct the segregation of dopaminergic axons into restricted compartments (Janis et al., 1999). Semaphorins also act as guidance cues for dopaminergic axons: Semaphorin3C (Sema3C) and Semaphorin3A (Sema3A) exert respectively an attractive effect and a growth promoting effect on dopaminergic axons (Hernández-Montiel et al., 2008). Finally, repulsive cues such as Slit-1 and Slit-2 guide ipsilateral growth of the dopaminergic axons and prevent midline crossing by a repulsive action from the midline where they are expressed (Dugan et al., 2011; Bagri et al., 2002; Lin et al., 2005). Therefore, a combination of cues expressed in different regions surrounding the mesostriatal pathway acts as spatiotemporal gradients during development, guiding the dopaminergic axonal navigation. Interestingly, some of these developmental cues are known to persist in the adult brain. For example, netrin- 1/Deleted in colorectal cancer (DCC) signalling is thought to contribute to the plasticity and remodelling of dopaminergic projection pathways (Osborne et al., 2005). However, very little is known about the expression of these cues in the lesioned brain and especially in the lesioned dopaminergic nigrostriatal pathway. Moreover, no studies reported so far the expression of intrinsic axon guidance molecules after cell therapy in animal models of PD.

As we previously showed that intranigral transplantation of VM fetal cells ensures a functional and anatomical repair in a mouse model of PD (Gaillard et al., 2009), we investigated here mRNA expression of axon guidance molecules in five different regions in the vicinity of the nigrostriatal pathway, before and after lesion and transplantation. We observed variations of semaphorin expression and at a lesser extent, modulation of Slit and ephrin mRNA levels. These findings suggest a role for these axon guidance molecules in appropriate wiring of transplanted dopaminergic cells, which may be of critical value in cell transplantation strategies.

2 Material and methods

2.1 Animals

All animal procedures were performed in accordance with the guidelines of the French Agriculture and Forestry Ministry (decree 87849) and of the European Communities Council Directive (86/609/EEC). All efforts were made to reduce the number and suffering of animals.

Adult (4 to 6 months old) C57BL/6 wild type female mice (supplier: R. Janvier Le Genest-Saint Isles, France) were used for lesion and transplantation procedures. Transgenic mice E12.5 embryos (B6D2F2 strain; provider: Dr. K. Kobayashi, Fukushima Medical University School of Medicine; Matsushita et al., 2002) overexpressing enhanced green fluorescent protein (EGFP) under the control of the tyrosine-hydroxylase promoter were used as a source of VM fetal cells for transplantation. For timed pregnancy, the day of the vaginal plug was considered as E0.5.

2.2 Lesion and transplantation

Chemical lesion of the SN pars compacta and cell transplantation were performed as previously described (Gaillard et al., 2009) with minor modifications. Briefly, mice were anesthetized by an intraperitoneal injection of Avertin (250 mg/kg). Then, 1 μl of 6-OHDA (8 μg/μl, Sigma #H-116) dissolved in sterile saline solution containing 0.1% ascorbic acid, was injected into the left SN pars compacta using a Hamilton syringe at the following coordinates AP: –3.2 mm (from bregma), L = 1.4 mm (from midline), V = 3.8 mm (from dura). For transplantation procedure, VM fetal cells were prepared from EGFP E12.5 transgenic embryo VMs and dissociated in 0.6% glucose saline solution. Cell suspension (150,000 cells/1.5 μl), or only vehicle solution for sham- grafted groups, was injected in the SN seven days after the lesion.

2.3 Quantitative real-time RT-PCR (qRT-PCR)

All experiments were performed according to the MIQE Guidelines (Bustin et al., 2009).

2.3.1 Tissue dissection and RNA extraction

We previously described that three days after grafting, numerous grafted fibres can be identified outside the transplant and then closely followed the host nigrostriatal pathway to initiate reinnervation of the striatum from the 7th day after transplantation (Gaillard et al., 2009). In this work, we first tested the influence of the transplant on axon guidance mRNA expression one day after grafting, before the first fibres grow out of the graft. Second, we checked for variations of axon guidance mRNA expression on their arrival onto the striatum, i.e. seven days after grafting. We also tested the effects of the lesion on axon guidance mRNA expression, one and seven days after lesion of the SN, this latter stage corresponding to the time of transplantation. Finally, in order to discriminate the effects of the lesion from the effects of the transplantation, we measured mRNA levels of axon guidance molecules eight and fourteen days after the lesion in sham- grafted animals. Experimental design is summarized in Diagram 1.

For this, six adult mice per group were killed by dislocation one day (LSND1) or seven days (LSND7) after the lesion, and one day (LTSND1) or seven days (LTSND7) after transplantation. Two other groups of five lesioned sham-grafted animals were used. The first group was sacrificed one day after vehicle transplantation (LSND8) and the second group eight days after vehicle transplantation (LSND14). Eleven intact adult animals were used as controls. Brains were removed and immediately frozen on dry ice. Sagittal cryostat sections (200 μm thick) were used to extract five brain regions (CPu, NAcc, MFB, Thal, and VM) with a 1mm-diameter punch, except for the VM where a 2mm-diameter punch was used (Palkovits, 1973). Localization of the punches was verified using the adult mouse brain atlas (Franklin & Paxinos, 2008) after a Nissl staining of the punched brain sections (Fig. 1). Briefly, sections were incubated in cresyl violet for 15 min at 60°C and were dehydrated in successive baths of increasing concentration of ethanol solutions (70% - 80% - 90% ethanol). Differential staining was then carried out successively in 95% and 100% ethanol solutions. Finally, sections were cleared in toluene and covered with DePeX mounting medium.

The punched tissue samples were immediately homogenized in 800 μl of TRIzol® reagent (Life Technologies, USA), and stored at –80°C until RNA extraction procedure. Total RNA was extracted, for each animal separately, according to the manufacturer instructions (TRIzol®); RNase-free glycogen (0.2–08.4 μg/μl; Invitrogen, USA) was added as a carrier during precipitation with isopropanol. RNA concentration was determined by spectrophotometry and RNA quality was assessed by the 260 nm/280 nm optical density ratio and by 0.7% agarose gel electrophoresis. RNA electrophoresis did not show any degradation or genomic DNA contamination (Fig. 2).

2.3.2 Reverse transcription

1 μg of total RNA was reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Briefly, RNA was incubated with 2 μl random hexamer primers (600 μM) at 70°C for 5 min then immediately put on ice. The following reagents were added to a final volume of 20 μl: 4 μl Transcriptor Reverse Transcriptase Reaction Buffer (5x concentration: 250 mM Tris/HCL, 150 mM KCL, 40 mM MgCl₂, pH approx. 8.5 at 25°C), 2 μl Deoxynucleotide mix (10 mM of each dNTP), 0.5 μl Transcriptor Reverse Transcriptase (20 U/μl), 0.5 μl of Protector RNase Inhibitor (40 U/μl). The mix was then incubated at 25°C for 10 min to allow primers annealing: the cDNA synthesis occurred at 50°C for 60 min and the reaction was stopped at 85°C for 5 min. The cDNA product was diluted for a calculated final concentration of 10 ng/μl and used as a template for quantitative PCR.

2.3.3 SYBR Green qPCR

Quantitative PCR was used to test mRNA expression of nine receptors and eleven axon guidance ligands on punched tissues in the five experimental groups described above (LSND1, LSND7, LTSND1, LTSND7 and control; see 2.3.1 section). Moreover, expression levels of receptors and axon guidance ligands that were found to vary after fetal cell transplantation, were tested in lesioned sham-grafted animals (LSND8 and LSND14). Receptor expression was tested in the VM region, where cell somas are located. Ligand expression was more particularly studied in the four regions located in the vicinity of the nigrostriatal pathway. In order to detect any potential genomic contamination, appropriate primer pairs were designed to span intronic sequences or to cover exon-intron boundaries (as specified in Table 1). They were tested on newborn and adult mouse brain cDNAs. Using 2% agarose gel electrophoresis, we verified that each set of primers amplified a single fragment at the expected size (outlined in Table 1) and that there was no genomic DNA amplification (Fig. 3). Melting curves analysis showed a single peak was observed confirming that no genomic DNA was amplified for each primer pair in each of the five tested regions (Example for ephrin-A5 in NAcc in Fig. 4). The efficiency of the qPCR was tested by a standard curve generated by performing qPCR with serial dilutions of the template cDNA. Each reaction consisted of 5 μl Fast SYBR Green Master Mix (Applied Biosystems, Foster City CA, USA), forward and reverse primers (at a final concentration of 0.1 μM), 2 μl template cDNA (final concentration 2 ng/μl), and water to a final volume of 10 μl. Each reaction was done in duplicate in 96-well plates (MicroAmp fast optical reaction plate with barcode, Applied Biosystems). Negative controls containing water instead of cDNA were included in each plate to detect possible contamination. The thermal cycling conditions were performed according to the pre-specified parameters of the fast mode of the Applied Biosystems 7500 Fast Real-Time PCR cycler. Samples were incubated at 95°C for 20 sec and then cycled 40 times at: 95°C for 3 sec and 60°C for 30 sec. This was followed by a melting curve stage that consisted of 15 sec at 95°C then 60 sec at 60°C followed by 15 sec at 95°C then 15 sec at 60°C. The products were analysed by melting curve analysis and by gel electrophoresis to verify that a unique product at the right size was amplified. GAPDH was chosen as the internal control among the four housekeeping genes that were tested: Actin, 18SRNA, GAPDH and cyclophilinA. Indeed, GAPDH showed negligible variations at the different stages and cerebral regions that were tested. Moreover, GAPDH qPCR showed the best efficiency curve (109.6%; y = –3.11x + 21.23; R²= 0.9946). Data were expressed as the percentage of GAPDH: 100×2^- ΔCt, where ΔCt = Ct_{gene of interest} - Ct _GAPDH. Expression was tested in the five described punched brain regions, in five to eleven animals per experimental group.

2.4 Statistical analysis

We used Prism software (version 5.0d) for statistical analysis. Data were analysed by a non- parametric Kruskal-Wallis analysis followed by Dunn’s post-hoc test for pairwise multiple comparisons. Results are expressed as the median with interquartile range. P < 0.05 values were accepted as statisticallysignificant.

3 Results

3.1 Netrin-1 and DCC expression does not significantly vary after lesion and transplantation

DCC receptor expression did not show any significant variation in the VM (Fig. 5A) following lesion and transplantation; neither did netrin-1 ligand transcripts in this region (Fig. 5B), in the Thal (Fig. 5D), in the NAcc (Fig. 5E) and in the CPu (Fig. 5F). In the MFB region, where netrin- 1 was more expressed than in other regions (Fig. 6A), Kruskal-Wallis test showed a slight significant group effect on netrin-1 mRNA transcripts (H(4) = 10.03, p = 0.04) (Fig. 5C). Post hoc multiple comparison tests did not show any significant differences of expression between the different experimental groups.

3.2 EphA5 expression decreases after lesion; ephrin-A2 and ephrin-A3 expression is modulated after lesion and transplantation

EphA5 receptor expression showed a significant decrease in the VM one day after the lesion as shown with a Dunn’s post-hoc test (Fig. 7A; p < 0.05). EphA5 ligands ephrin-A2 (Fig. 7B-D) and ephrin-A3 (Fig. 7G-I) did not show any variation of expression in both mesencephalon and diencephalon. Moreover, lesion and transplantation did not modulate ephrin-A2 expression in the CPu (Fig. 7F), whereas it was found to vary in the NAcc region (Fig. 7E; Kruskal-Wallis test: H(4) = 15.80, p = 0.0033), with a significant decrease one day after lesion, and seven days after grafting in comparison to control. In sham-grafted animals, the expression was similar to controls eight and fourteen days after the lesion (Fig. 8A). Kruskal-Wallis test showed a slight significant group effect on ephrin-A3 mRNA transcripts in the NAcc (Fig. 7J; H(4) = 10.2, p = 0.0372), but no significant differences were observed after statistical post hoc multiple comparison test. Interestingly, ephrin-A3 mRNA expression gradually decreased in the CPu and showed a significant group effect (Fig. 7K; Kruskal-Wallis: H(4) = 22.07, p = 0.0002) and a significant decrease one day after transplantation in comparison to control and to the first day after lesion. Values of ephrin-A3 expression in sham-grafted animals were not different from control in the CPu (Fig. 8B). Ephrin-A5 transcripts did not vary significantly after lesion and transplantation in any of the five studied regions(Fig. 7L-P).

3.3 Expression of class-3 and class-7 semaphorins is widely modulated along the nigrostriatal pathway

As neuropilins are receptors for secreted class-3 semaphorins and associate to plexins to trigger intracellular signalling cascade, we tested the expression of neuropilin 1 (Nrp1), neuropilin 2 (Nrp2) and their molecular partners plexin-A2 (PlxnA2) and plexin-A4 (PlxnA4) in the VM. Nrp1, Nrp2 as well as PlxnA2 semaphorin receptors expression did not vary in the VM region after lesion or transplantation (Fig. 9A, B, C). PlxnA4 expression was stable in the VM region following lesion, and significantly decreased one day after transplantation compared to control (Fig. 9D; Kruskal-Wallis: H(4) = 12.06, p = 0.0169) and to sham-grafted animals (Fig. 8C). Seven days after grafting, expression tended to increase and to reach control level (Fig. 9D). Sema3A and Sema3F expression significantly varied exclusively in the CPu, where their basal expression was found to be higher than in other tested regions (Fig. 6E, G). Sema3A expression decreased seven days after lesion (Kruskal-Wallis: H(4) = 15, p = 0.0047), and was restored to control levels after transplantation (Fig. 10E). Moreover, in sham-grafted animals Sema3A expression was not different from control (Fig. 8D). Sema3F transcripts were 1.5 times less abundant one day after grafting than six days later (Fig. 10O; Kruskal-Wallis: H(4) = 14.95, p = 0.0048; Dunn’s test: p < 0.01) whereas its expression did not vary in sham-grafted animals (Fig. 8E). Sema3C expression slightly varied (Kruskal-Wallis: H(4) = 10.24, p = 0.0366) but multiple comparison statistics did not show any significant differences (Fig. 10J). In the four other regions, Sema3A (Fig. 10A-D), Sema3C (Fig. 10F-I) and Sema3F (Fig. 10K-N) did not show significant variations upon lesion or transplantation. Finally, Sema7A showed significant variations in four out of five tested regions: in the MFB, a progressive decrease was produced upon lesion that reached a lower limit (1.3 fold decrease) after seven days. Following that time period Sema7A transcripts increased progressively upon transplantation (Fig. 11B; Kruskal-Wallis: H(4) = 10.94, p = 0.0273) and in sham-grafted animals (Fig. 8F). Sema7A mRNAs decreased 1.7 times in the Thal, one day and seven days after lesion as compared to control levels (Fig. 11C; Kruskal-Wallis: H(4) = 14.71, p = 0.0054; Dunn’s test: p < 0.05 and p < 0.01 respectively). Then, it progressively increased after transplantation to reach control values whereas its expression remained lower from that of control in sham-grafted animals (Fig. 8G, LSND14). In the NAcc, the lesion induced a slight non-significant decrease of expression, that was 1.8 times lower than expression seven days after grafting (Fig. 11D; Kruskal-Wallis: H(4) = 13.88, p = 0.0077; Dunn’s test: p < 0.05). In sham-grafted animals, we detected a decrease of expression fourteen days after the lesion in comparison to control (Fig. 8H, LSND14). In the CPu, Sema7A mRNA expression decreased 1.5 times, one day after transplantation in comparison to control. This level of expression was restored to control values seven days after transplantation (Fig. 11E; Kruskal-Wallis: H(4) = 9.996, p = 0.0405; Dunn’s test: p < 0.05) whereas it remained lower to that of control in sham-grafted animals (Fig. 8I, LSND14). Finally, in the VM, we observed the same tendency of variation (Fig. 11A; Kruskal-Wallis: H(4) = 11.57, p = 0.0209), although post hoc multiple comparison tests did not reveal any significant differences of expression between any groups.

3.4 Expression of Slit-3, Robo1 and Robo2 decreases after lesion and is restored later on independently from transplantation

Variations of expression of Robo1 and Robo2 receptors were detected respectively in the VM and in the MFB. More particularly, in the VM, Robo1 levels increased 1.5 times seven days after grafting compared to its expression level one day after the lesion (Fig. 12A; Kruskal-Wallis test: H(4) = 14.03, p = 0.0072; Dunn’s test: p < 0.01). Its expression was not different from that of control in sham-grafted animals (Fig. 8J). Robo2 transcripts displayed a 2.5-fold decrease after lesion in the MFB in comparison to control (Dunn’s test: p < 0.001). This low level of expression increased progressively to reach control values seven days after transplantation (Fig. 12B; Kruskal-Wallis test: H(4) = 20.15, p = 0.0005) as also observed in sham-grafted animals (Fig. 8K). Robo3 expression was not modulated by lesion or transplantation (Fig. 12C). Slit-1 and Slit-2 ligand expression did not vary in our model in the five tested regions (Fig. 12D-M), whereas Slit-3 transcript expression varied exclusively in the Thal and in the NAcc: in the Thal, a 2.2 fold decrease was measured one day after the lesion (Dunn’s test: p < 0.05), and was restored to control levels seven days after grafting (Fig. 12P, Kruskal-Wallis test: H(4) = 16.93, p = 0.002). Expression also reached control levels in sham-grafted animals (Fig. 8L). In the NAcc, a gradual decrease of Slit-3 mRNA expression was induced by the lesion and reached a 1.7-fold decrease after seven days compared to control levels (Fig. 12Q; Kruskal-Wallis test: H(4) = 10.90, p = 0.0277; Dunn’s test: p < 0.05). After transplantation, the level of expression was not different from that of control (Fig. 12Q) whereas it was significantly lower after one day in sham-grafted animals (Fig. 8M, LSND8).

Significant variations of axon guidance molecule expression in comparison to controls are summarized in Table 2.

4 Discussion

This work shows for the first time that expression of specific axon guidance molecules is modulated after degeneration and repair of the nigrostriatal pathway in a mouse model of PD.

As summarized in Table 2, the expression of three out of four main axon guidance families is modified by lesion and/or transplantation of VM fetal cells: the lesion mainly affects expression of Robo/Slit members, whereas both lesion and transplantation modify Eph/ephrin and Plxn/Sema mRNA levels. The lesion induces a decrease of expression, whereas there are distinct effects of the transplantation: ephrinsexpression is diminished after grafting whereas Sema7A expression is increased.

4.1 DCC and netrin-1 may not be involved in the reconstruction of the nigrostriatal pathway

We did not find any effect of lesion or transplantation on DCC receptor mRNA expression in the VM, although this receptor has been localized in the ventral tier of the adult SN pars compacta (Osborne et al., 2005). Nevertheless, DCC is also expressed in dopaminergic neurons of the ventral tegmental area and around the interfascicular nucleus in the adult (Osborne et al., 2005). Thus, measuring its expression in the VM (that include SN and ventral tegmental area) does not reflect its involvement in the nigrostriatal circuitry solely.

Moreover, netrin-1 transcripts did not show any significant variations in the different experimental procedures implemented here, suggesting that this molecule is not affected by loss of SN dopaminergic neurons or by the presence of grafted fetal cells. Indeed, in the adult, netrin- 1 expression is limited to cholinergic interneurons in the dorsal striatum (Schatzmiller et al., 2008), meaning that it is weakly represented in this target region of nigrostriatal projections. Consequently, netrin-1 may not be a major actor in the repair of the nigrostriatal pathway using VM fetal cells and this could be also the case for grafts using embryonic stem cell-derived dopaminergic neurons. Indeed, it has been previously shown that neurite outgrowth of embryonic stem cell-derived dopaminergic neurons expressing DCC is not directed by netrin-1 when co- cultured with netrin-1 producing cells (Lin & Isacson, 2006). Taken together, these set of findings indicate that netrin-1 alone may not be sufficient to support axon guidance in transplantation paradigms but should probably be associated with other factors such as glial cell line-derived neurotrophic factor (GDNF) (Zhang et al., 2013).

4.2 Robo-Slit molecules may be weakly involved in the anatomical repair of the nigrostriatal pathway

Although Robo1 and Robo2 receptors are expressed in the adult SN (Marillat et al., 2002), we did not observe a significant decrease of expression of these receptors after lesion of the SN dopaminergic neurons (data not shown for Robo2), suggesting that these receptors are more likely to be expressed in GABAergic neurons of the SN pars reticulata. Moreover, Robo2 transcripts displayed a two-fold decrease after lesion in the MFB, suggesting that Robo2 is expressed in other MFB fibres than the dopaminergic axons arising from the SN such as dopaminergic axons growing out from the ventral tegmental area, the serotoninergic fibres from the raphe nuclei, or the noradrenergic fibres from the locus coeruleus.

Among the tested slit molecules, only Slit-3 expression level was modified and decreased by the lesion. This was especially the case in the NAcc and in the Thal, where Slit-3 is known to be expressed during embryogenesis (Marillat et al., 2002) and in the intact adult brain as described in the present study (Fig. 6K). This indicates that the loss of nigrostriatal dopaminergic fibres could influence the expression of Slit-3 in others cell populations in the NAcc and the Thal.

Interestingly, the presence of grafted cells restored a level of expression similar to that of control in the NAcc from the first day after transplantation, whereas Slit-3 expression remained low in sham-grafted animals and increased six days later. This suggests that the presence of grafted cells may accelerate the restoration of the expression to the control level.

4.3 Ephrin-A2 may have an inhibitory role in the early connection of grafted axons

We reported a two-fold decrease in EphA5 receptor expression in the VM after lesion, confirming the presence of this receptor in dopaminergic cells of the adult SN pars compacta (Deschamps et al., 2009; Cooper et al., 2009). More precisely, we previously showed that EphA5 is expressed in this region from E13.5 in the rodent developing nigrostriatal pathway (Deschamps et al., 2009). Thus, fetal cells collected at E12.5 do not express this receptor at the time of transplantation and neither later, since EphA5 expression was not restored in the VM after transplantation. Furthermore, ephrin-A5 did not show any significant variation of expression after lesion or transplantation although it is expressed in the vicinity of the intact nigrostriatal pathway (Fig. 6D; Deschamps et al., 2010). This indicates that ephrin-A5-EphA5 couple may be only weakly involved during the first seven days of the nigrostriatal repair. Among other potential ligands of EphA5, striatal ephrin-A3 expression was transiently maintained to lower levels of expression than in control and sham-grafted animals one day aftertransplantation. This suggests that the presence of grafted cells could inhibit ephrin-A3 expression in the CPu. A similar but more extended modulation was observed for ephrin-A2. Ephrin-A2 transcripts were decreased in the NAcc one day after SN lesion and this low expression was reinforced seven days after transplantation. As expression in sham-grafted animals was similar to control levels, this suggests that grafted cells could diminish the expression of ephrin-A2 in the NAcc. This could create a permissive environment to reconnect the dopaminergic fibres as this molecule has been shown to have repulsive effects on dopaminergic axons during development (Janis et al., 1999) and in other systems such as the regeneration of lesioned optic nerve in adult (Symonds et al., 2007).

4.4 Sema7A may be involved in the repair of the nigrostriatal pathway after grafting

Among the four semaphorin receptors and co-receptors that were tested, PlxnA4 expression was the only one to be modified by the graft, showing that the presence of grafted cells induced a reduction of expression of this receptor in the VM that was restored to control levels six days later. This indicates that the presence of grafted cells can modify axon guidance molecule expression in the host adult tissue. Surprisingly, the lesion and/or transplantation slightly affected the expression of class-3 semaphorins: whereas the expression of Sema3F was not different from that of control; the lesion specifically affected the level of Sema3A in the CPu, where it is basally more expressed (from 1.1 to 4.8 times) than in the four other tested regions (Fig. 6E, G). Indeed, loss of dopaminergic projections induced a significant decrease of Sema3A in the CPu seven days after the lesion. Its expression was restored after eight and fourteen days independently from the presence of grafted cells. This could indicate either that Sema3A is not modulated by the presence of dopaminergic fibres (which is unlikely since the loss of dopaminergic fibres decreased its expression), or that Sema3A may have a role in the refinement of the connections, i.e. later than seven days after the transplantation. In this case, Sema3A could induce positive signals for the reconstruction of the pathway as previously shown in vivo and in vitro (Diaz-Martinez et al., 2013; Tamariz et al., 2011).

Unlike class-3 semaphorins, Sema7A expression was widely modulated after lesion and/or transplantation in our animal model of PD: the SN lesion decreased Sema7A expression in the MFB and Thal, transplantation increased its expression in the Thal, NAcc and CPu, whereas no effect of the lesion was observed in the VM. These observations suggest that this membrane- anchored semaphorin is expressed on cells communicating with the SN dopaminergic neurons. Increase of expression of Sema7A in the Thal, NAcc and CPu during the reconstruction of the nigrostriatal pathway seven days after grafting may help to guide axons to their final targets by enhancing axon growth and/or promoting axon tract formation as it has been described during embryonic development. This could be achieved through b1-integrin receptors and activation of MAPK signalling pathways as it has been outlined for this semaphorin (Pasterkamp et al., 2003). Studying guidance of the nigrostriatal pathway in Sema7A knock-out mice may help to understand the spatiotemporal involvement of this molecule in the restoration of this pathway. Another interesting point is the implication of Sema7A in the inflammatory response. Indeed, Sema7A is known to stimulate cytokine production and to be critical for the inflammatory immune response (Suzuki et al., 2007). Knowing that 6-OHDA lesion in mice induces inflammatory response during the first week after lesion (Haas et al., 2016), it would be interesting to discriminate the role of this molecule in axon growth and tract formation from its role in the inflammatory responses in our model of Parkinson’s disease.

5 Conclusion

This work shows for the first time that axon guidance molecules, known to be involved in the establishment of the nigrostriatal pathway during development (see review by Prestoz et al., 2012), are modulated by transplantation of fetal VM cells in a mouse model of PD.

More particularly, we propose that Sema7A is necessary for the topographic reconstruction of the nigrostriatal pathway. Its increased expression after cell transplantation in the Thal, NAcc and CPu may help axons to connect to their appropriate targets together with the decrease of expression of ephrin-A2 in the NAcc that could create permissive environment for the path of dopaminergic axons (Fig. 13). These molecules should be considered if we aim to improve cell therapy efficiency.

Finally, to improve the efficiency of cell therapy in Parkinson’s patients, there is a need to characterize the molecular identity of the cells to be grafted and more particularly the expression of axon guidance molecules and their receptors. As other sources of cells for dopaminergic neuron replacement are currently tested (Astradsson & Aziz, 2016; Lindvall, 2015), such as embryonic stem cell-derived dopaminergic neurons or dopaminergic neurons obtained from somatic cells of patients (see review by Kang et al., 2016), it would be beneficial to compare their molecular identity and the involvement of axon guidance molecules after grafting of these different cell types to determine the most efficient way to repair the nigrostriatal pathway.

Footnotes

Acknowledgments

This work was supported by the Fondation de France, the INSERM, the University of Poitiers, the Lebanese University, Région Poitou-Charentes and FEDER European program.

Authors certify to have no conflict of interest.

References

Astradsson

, & Aziz

(2016). Parkinson’s disease: Fetal cell or stem cell derived treatments. BMJ, 7, h6340.

Bagri

, Marín

, Plump

A.S.

, Mak

, Pleasure

S.J.

, Rubenstein

J.L.R.

, & Tessier-Lavigne

(2002). Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron, 33, 233–248.

Bustin

S.A.

, Benes

, Garson

J.A.

, Hellemans

, Huggett

, Kubista

, Mueller

, Nolan

, Pfaffl

M.W.

, Shipley

G.L.

, Vandesompele

, & Wittwer

C.T.

(2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611–622.

Cooper

M.A.

, Kobayashi

, & Zhou

(2009). Ephrin-A5 regulates the formation of the ascending midbrain dopaminergic pathways. Developmental Neurobiology, 69, 36–46.

Deschamps

, Faideau

, Jaber

, Gaillard

, & Prestoz

(2009). Expression of ephrinA5 during development and potential involvement in the guidance of the mesostriatal pathway. Experimental Neurology, 219, 466–480.

Deschamps

, Morel

, Janet

, Page

, Jaber

, Gaillard

, & Prestoz

(2010). EphrinA5 protein distribution in the developing mouse brain. BMC Neuroscience, 25, 105–121.

Desplats

, Lee

H-J.

, Bae

E-J.

, Patrick

, Rockenstein

, Crews

, Spencer

, Masliah

, & Lee

S.-J.

(2009). Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proceedings of the National Academy of Sciences of the United States of America, 106, 13010–13015.

Díaz-Martínez

N.E.

, Tamariz

, Díaz

N.F.

, García-Peña

C.M.

, Varela-Echavarría

, & Velasco

(2013). Recovery from experimental parkinsonism by semaphorin-guided axonal growth of grafted dopamine neurons. Molecular Therapy, 21, 1579–1591.

Dugan

J.P.

, Stratton

, Riley

H.P.

, Farmer

W.T.

, & Mastick

G.S.

(2011). Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals. Molecular and Cellular Neuroscience, 46, 347–356.

10.

Franklin

K.B.J.

, & Paxinos

(2008). The mouse brain in stereotaxic coordinates. Third edition. New York: Academic Press.

11.

Gaillard

, Decressac

, Frappé

, Fernagut

P.O.

, Prestoz

, Besnard

, & Jaber

(2009). Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants. Neurobiology of Disease, 35, 477–488.

12.

Haas

S.J-P.

, Zhou

, Machado

, Wree

, Krieglstein

, & Spittau

(2016). Expression of Tgfβ1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson’s Disease. Frontiers in Molecular Neuroscience, 9, article 7.

13.

Hernández-Montiel

H.L.

, Tamariz

, Sandoval-Minero

M.T.

, & Varela-Echavarría

(2008). Semaphorins 3A, 3C, and 3F in mesencephalic dopaminergic pathfinding. Journal of Comparative Neurology, 506, 387–397.

14.

Janis

L.S.

, Cassidy

R.M.

, & Kromer

L.F.

(1999). Ephrin-A binding and EphA receptor expression delineate the matrix compartment of the striatum. Journal of Neuroscience, 19, 4962–4971.

15.

Kang

J.F.

, Tang

B.S.

, & Guo

J.F.

(2016). The Progress of Induced Pluripotent Stem Cells as Models of Parkinson’s Disease. Stem Cells International, 2016, 4126214.

16.

, Duarte

, Kocabas

, Works

, McConnell

S.K.

, & Hynes

M.A.

(2014). Evidence for topographic guidance of dopaminergic axons by differential Netrin-1 expression in the striatum. Molecular and Cellular Neuroscience, 61, 85–96.

17.

Lin

, Rao

, & Isacson

(2005). Netrin-1 and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons. Molecular and Celluylar Neuroscience, 28, 547–555.

18.

Lin

, & Isacson

(2006). Axonal growth regulation of fetal and embryonic stem cell-derived dopaminergic neurons by Netrin-1 and Slits. Stem Cells, 24, 2504–2513.

19.

Lindvall

(2015). Treatment of Parkinson’s disease using cell transplantation. Philosophical Transactions of the Royal Society London B Biological Sciences, 370, 20140370.

20.

Marillat

, Cases

, Nguyen-Ba-Charvet

K.T.

, Tessier-Lavigne

, Sotelo

, & Chédotal

(2002). Spatiotemporal expression patterns of slit and robo genes in the rat brain. Journal of Comparative Neurology, 442, 130–155.

21.

Matsushita

, Okada

, Yasoshima

, Takahashi

, Kiuchi

, & Kobayashi

(2002). Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development. Journal of Neurochemistry, 82, 295–304.

22.

Miocinovic

, Somayajula

, Chitnis

, & Vitek

J.L.

(2013). History, applications, and mechanisms of deep brain stimulation. JAMA Neurology, 70, 163–171.

23.

Obeso

J.A.

, Rodriguez-Oroz

M.C.

, Goetz

C.G.

, Marin

, Kordower

J.H.

, Rodriguez

, Hirsch

E.C.

, Farrer

, Schapira

A.H.V.

, & Halliday

(2010). Missing pieces in the Parkinson’s disease puzzle. Nature Medecine, 16, 653–661.

24.

Osborne

P.B.

, Halliday

G.M.

, Cooper

H.M.

, & Keast

J.R.

(2005). Localization of immunoreactivity for deleted in colorectal cancer (DCC), the receptor for the guidance factor netrin-1, in ventral tier dopamine projection pathways in adult rodents. Neuroscience, 131, 671–681.

25.

Palkovits

(1973). Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Research, 14, 449–450.

26.

Pasterkamp

R.J.

, Peschon

J.J.

, Spriggs

M.K.

, & Kolodkin

A.L.

(2003). Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature, 424, 398–405.

27.

Prestoz

, Jaber

, & Gaillard

(2012). Dopaminergic axon guidance: Which makes what? Frontiers in Cellular Neuroscience, 31, 1–13.

28.

Shatzmiller

R.A.

, Goldman

J.S.

, Simard-Emond

, Rymar

, Manitt

, Sadikot

A.F.

, & Kennedy

T.E.

(2008). Graded expression of netrin-1 by specific neuronal subtypes in the adult mammalian striatum. Neuroscience, 157, 621–636.

29.

Suzuki

, Okuno

, Yamamoto

, Pasterkamp

R.J.

, Takegahara

, Takamatsu

, Kitao

, Takagi

, Rennert

P.D.

, Kolodkin

A.L.

, Kumanogoh

, & Kikutani

(2007). Semaphorin 7A initiates T-cell-mediated inflammatory responses through a1b1 integrin. Nature, 446, 680–684.

30.

Symonds

A.C.

, King

C.E.

, Bartlett

C.A.

, Sauvé

, Lund

R.D.

, Beazley

L.D.

, Dunlop

S.A.

, & Rodger

(2007). EphA5 and ephrin-A2 expression during optic nerve regeneration: A ‘two- edged sword’. European Journal of Neuroscience, 25, 744–752.

31.

Tamariz

, Wan

A.C.A.

, Pek

Y.S.

, Giordano

, Hernández-Padrón

, Varela-Echavarría

, Velasco

, & Castaño

V.M.

(2011). Delivery of chemotropic proteins and improvement of dopaminergic neuron outgrowth through a thixotropic hybrid nano-gel. Journal of Materials Science: Materials in Medicine, 22, 2097–2109.

32.

Thompson

L.H.

, Grealish

, Kirik

, & Björklund

(2009). Reconstruction of the nigrostriatal dopamine pathway in the adult mouse brain. European Journal of Neuroscience, 30, 625–638.

33.

Worth

P.F.

(2013). How to treat Parkinson’s disease in. Clinical Medicine, 13, 93–96.

34.

Zhang

, Jin

, Ziemba

K.S.

, Fletcher

A.M.

, Ghosh

, Truit

, Yurek

D.M.

, & Smith

G.M.

(2013). Long distance directional growth of dopaminergic axons along pathways of netrin-1 and GDNF. Experimental Neurology, 250, 156–164.