Introduction
The progressive degeneration of nigral dopamine neurons is a central element in the pathophysiology of Parkinson’s disease (PD). While no protective therapy has been identified, dopaminergic drugs offer a symptomatic relief to patients but over time these medications become less effective as the disease progresses. Over the last three decades, many studies have shown that replacement of lost dopamine neurons by transplantation of dopamine-producing cells into the diseased rodent brain is a promising avenue of research for PD [
3,
4]. Among the various sources of cells tested to date, fetal dopaminergic progenitors derived from the developing ventral mesencephalon (VM) remain the best source of cells identified to date. These cells have entered clinical trials, and while the results are variable, it has been shown that in some patients there is substantial long-term benefit from such grafts [
4,
19,
25,
32,
42,
58]. While the reported long-term clinical improvement seen in some patients clearly sustain the therapeutic potential of this approach, the mixed outcome of these trials has underscored the importance of patient selection, the need for a better standardization of the surgical procedure and optimization of the trial design [
3].
These clinical studies have also underlined the limited predictability of the animal models of PD that were available at the time as well as the existence of additional factors which may have influenced the final clinical outcome [
44]. All the pre-clinical studies performed prior to the clinical trials were conducted in toxin-based models of PD obtained by either injection of 6-hydroxydopamine (6-OHDA) in rats or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in non-human primates [
4‐
6,
8,
51]. Although these models are useful for looking at the restoration of the dopaminergic striatal innervation, they fail to replicate important pathological features including the development of the alpha-synuclein (α-synuclein) aggregation seen in the PD brain, which may influence the integration and long-term survival of the transplanted cells. In addition, neurotoxic lesions are acute ways of damaging the dopaminergic system whilst the PD pathology is progressive. Finally, most rodent studies utilize isogenic grafts (i.e. tissue from inbred strains) while patients have received allografted tissue, thereby making the immune response an important modifier of the clinical outcome.
In order to overcome some of the limitations of the neurotoxin models of PD, attempts have been made to create new models which replicate the progressive nature of the pathology and its α-synuclein basis [
55], but this has not really been done to any great extent to look at the survival, integration and functional impact of fetal VM dopaminergic grafts. The relevance of further research in this area is supported by the recent observations of Lewy body-like pathology in donor-derived dopamine neurons grafted into subjects with PD, suggesting a mechanism of host-to-graft disease propagation [
28,
31] and thereby demonstrating that the α-synuclein pathology of the host brain can affect the grafted cells themselves. In vitro studies provided the first evidence for the existence of a “prion-like” propagation of aggregated pathogenic α-synuclein, which was later observed in rodents and non-human primates [
18,
33‐
35,
45,
57]. The recent development of a rodent model of PD based on targeted overexpression of human α-synuclein using intra-nigral injection of an adeno-associated viral vector (AAV) has opened the possibility to investigate in more detail the graft effects in the host (as opposed to the host on the graft) and have done so by conducting side-by-side comparative studies with the standard 6-OHDA-induced rat model of PD.
Here we demonstrate that such grafts induce a sustained low-grade level of inflammation and that this immune response negatively impacts on the survival of the grafted neurons. Furthermore we provide evidence that immunosuppression using cyclosporin A (CsA) not only promoted the long-term survival of the transplanted cells but also afforded a restorative effect in several different models of PD based on α-synuclein or MPTP toxicity. This therapeutic effect resulted from a combination of non-cell autonomous and cell autonomous mechanisms, and not just through its immunosuppressive effects.
Material and methods
Primary cultures of cortical neurons
Primary neuronal cultures were prepared from the E16 C57BL/6 mouse brain. Dissociated cortical neurons were plated onto 6-well plates at a density of 50,000 cells/cm2. Treatment with CsA (10 μM) was performed at 7–9 days in vitro and analyses were carried out 24 h later.
Viral vectors
Production of the AAV2/6 vector expressing the human wild-type α-synuclein under the control of the human synapsin-1 promoter and enhanced using a woodchuck hepatitis virus post-transcriptional regulatory element was performed as previously described (Decressac et al. 2012). Genome copy (gc) titer was determined by real-time quantitative PCR and the following vector concentration was used: 1.6 × 1012 gc/ml.
The Cre-regulated AAV-α-synuclein vector (thereafter referred as AAV-FlexOFF-α-synuclein) was generated by inserting the cDNA of wild-type human α-synuclein between two pairs of heterotypic, antiparallel loxP sequences [
2]. This vector design results in flipping and inactivation of the transgene specifically in Cre-expressing cells. Validation of the inactivation of the transgene in dopamine neurons was confirmed by injection of the AAV vector (6.8 × 10
9 gc/ml) in the ventral midbrain of Dat-Cre mice (Additional file
1: Supplementary Figure 1) and a similar dose was used for experiments in Dat-Cre
ERT2 mice.
Animals
Three-month-old, adult female Sprague Dawley rats (Charles River), 225 − 250 g at the time of surgery, were housed two to three per cage. Adult C57Bl/6, DAT-IRES-Cre (stock number 006660, Jackson Laboratories) and DAT-CreERT2 mice (stock number 016583, Jackson Laboratories), aged 12 weeks old at the time of AAV virus injection, were housed two to six per cage. In both Cre transgenic mice, expression of the Cre recombinase is under control of the promoter of the dopamine transporter (DAT) and hence is detected in dopaminergic neurons since embryonic stage. While the enzyme can freely translocate to the nucleus to exert its activity in DAT-IRES-Cre mice, its function is conditioned by exposure to synthetic ligands such as tamoxifen in the DAT-CreERT2 mice since the enzyme is fused to a mutant variant of the human estrogen receptor.
Ten-month-old transgenic mice overexpressing wild-type human α-synuclein under the neuron-specific Thy1 promoter (“Line 61”; provided by Pr. E. Masliah, University of California) were housed under similar conditions [
48]. All animals had
ad libitum access to food and water during a 12 h light/dark cycle.
All procedures were conducted in accordance with guidelines set by the local Ethical Committees for the use of laboratory animals (Lund-Malmo region and Naples), the European Directives (2010/63/EU), and the federal guidelines of the Public Health Service Policy on the Humane Care and Use of Laboratory Animals.
Lesion procedures
In rats, all surgical procedures were performed under general anesthesia using a 20:1 mixture of fentanylcitrate (Fentanyl) and medetomidin hypochloride (Dormitor) (Apoteksbolaget, Sweden), injected intraperitoneally (i.p). Solutions containing the AAV-α-synuclein vector or 6-OHDA were injected using a 10 μl Hamilton Neuros syringe. Rats received either 3 μl of AAV-α-synuclein into the substantia nigra (SN) or 3 μl of 6-OHDA (3.5 μg/μl free base dissolved in a solution of 0.2 mg/ml L-ascorbic acid in 0.9 % w/v NaCl) into the medial forebrain bundle (MFB). Infusions were performed at a rate of 0.2 μl/min and the needle was left in place for an additional 3 min period before being slowly retracted. Injections were carried out unilaterally on the right side, at the following coordinates (flat skull position) for the SN: antero-posterior: −5.3 mm, medio-lateral: −1.7 mm, dorso-ventral: −7.2 mm below dural surface; and for the MFB: antero-posterior: −4.4 mm, medio-lateral: −1.1 mm, dorso-ventral: −7.8 mm below dural surface, calculated relative to bregma according to the stereotaxic atlas of Paxinos and Watson [
43].
In mice, surgical procedures were performed under general anesthesia using isoflurane. A 5 μl Hamilton syringe was used to inject 1 μl of AAV vector in the mouse SN at the following coordinates: antero-posterior: −2.7 mm, medio-lateral: −1.2 mm, dorso-ventral: −4.2 mm below dural surface.
Tamoxifen administration
Tamoxifen (Sigma, T-5648) was dissolved in corn oil (Sigma, C-8267) and ethanol in a 9:1 mixture at a final concentration of 10 mg/ml. New mixture was prepared every other day.
Eight weeks after AAV vector injection, Dat-CreERT2 mice were tested for motor performance and manifest animals received i.p injections of 2 mg of tamoxifen per day for 5 consecutive days to inactivate the transgene in dopamine neurons.
Transplantation procedure
VM from E14 rat embryos were dissected as previously described [
10], incubated in Dulbecco’s modified eagle medium (DMEM, Invitrogen) containing 0.1 % trypsin (Sigma-Aldrich) and 0.05 % DNase (Sigma-Aldrich) for 20 min at 37 °C and mechanically dissociated into a single cell suspension. After centrifugation (500
g, 5 min, 4 °C), the number of viable cells was estimated by trypan blue staining (Sigma-Aldrich). Cells were then re-suspended in DMEM/DNase at a concentration of 150,000 cells/μl and kept at room temperature until grafted.
Transplantation was performed under general anesthesia as described above. About 150,000 cells were injected as two 0.75 μl deposits in the lesioned striatum at the following coordinates: antero-posterior: +0.6 mm, medio-lateral: −3.1 mm, dorso-ventral: −4.5/-3.5 mm below the dural surface. Rats assigned to the sham control group received a similar injection of the vehicle solution only. Leftover cells were subject to an estimation of cell viability using trypan blue exclusion and survival was >95 %.
Cyclosporin a treatment
For the transplantation experiments, rats received daily i.p injections of cyclosporin A (CsA) (10 mg/kg) or vehicle solution starting 2 days before transplantation and until the endpoint of the experiment [
8,
21].
For the experiments in the AAV-FlexOFF-α-synuclein mouse model, animals received i.p injections of CsA (20 mg/kg, Sigma-Aldrich) every other day from the day following the last injection of tamoxifen and until the time of sacrifice.
For the experiments in the Thy1-α-synuclein mice, 10-month-old transgenic mice and wild-type littermates received daily i.p injections of CsA (20 mg/kg) or vehicle solution for 6 weeks.
The dose of CsA used in mice was calculated as follow, based on the standard dose used for the rats [
30] and Body Surface Area (BSA) conversion (values from FDA Draft guidelines): dose in mice (mg/kg) = dose in rats (mg/kg) × (mouse Km/rat Km), where K
m factor = Weight (kg)/Body Surface area (m
2); for mice: weight = 0.02 kg, BSA = 0.007 and K
m = 3; for rats: weight = 0.15 kg, BSA = 0.025 and K
m = 6. All behavioural tests were performed at least 2 h after CsA injection.
Protocols for MPTP and CsA injections
C57BL/6 J mice (Jackson Laboratories, Bar Harbor, ME, USA) (8 weeks old) were injected with increasing doses of MPTP (8 mg/kg, 10 mg/kg, 24 mg/kg and 32 mg/kg, 5 days/week, i.p.) for a total of 4 weeks (30). Three days following MPTP, mice were administered either vehicle or CsA at a concentration of 20 mg/kg in normal saline for 4 weeks (5 days /week, i.p.).
Estimation of transplants volume
The volume of the graft was calculated from TH-stained striatal sections using the Cavalieri formula taking into account the sum of all the areas and correcting for section thickness and sample frequency [Volume (mm
3) = Sum of areas (mm
2) × 35 μm × 6 series] [
30].
ELISA analysis
Striata were dissected and homogenized in TPER lysis buffer containing phosphatase cocktail I and II (1:100; Sigma-Aldrich). Samples were analyzed for the level of cytokines (Pierce, ThermoFischer Scientific).
Statistical analysis
Statistical analyses were conducted using the GraphPad Prism software (version 6.0f) or the Jmp 11 software (SAS). Unpaired two-tailed Student's t tests or one-way ANOVA tests with Dunnett's multiple comparison test were performed to analyze the difference between experimental and control groups. Two-way repeated measures ANOVA tests were used to detect interactions between time and treatment. The data were collected and processed in a randomized and blinded manner. No statistical methods were used to predetermine sample size, but our sample sizes are similar to those generally employed in the field. All values are presented as mean ± standard error of the mean. Statistical significance was set at P < 0.05.
Behavioural tests, histological procedures, cell counting, optical densitometry analysis and western blot protocols are described in details in Additional file
2: Supplementary Information.
Discussion
Preclinical studies looking at therapeutic strategies in novel disease models may not only better inform their translation to the clinic, but also opens the possibility of unraveling novel pathological mechanisms which may have clinical implications in their own right. In the present study, we examined the behaviour of VM cell transplants in the recently developed AAV-α-synuclein based model of PD in comparison with the standard 6-OHDA-induced model. Although functional recovery was observed in both models, this set of experiments revealed that factors specific to the AAV-based models negatively impacted on the survival of the transplanted dopaminergic cells. Notably, we focused our attention on the immune and inflammatory reaction as (
i) this deleterious process occurs in the human PD brain and is likely to contribute to the progression of the disease [
14,
37]; (
ii) as seen in rodent models [
24,
50], a sustained immune response was reported in PD patients who underwent cell transplantation of fetal mesencephalic cells [
28] and (iii) the use of immunosuppressive drugs may have influenced graft survival and efficacy in clinical trials in PD [
19,
32,
42,
58]. Despite this knowledge and the well-documented pro-survival effect of CsA on dopamine progenitors grafted in the 6-OHDA model of PD [
7,
11], the use of immunosuppression in the context of cell therapy in for PD patients has not been studied. In the open-label study, which demonstrated clinical benefit, patients were given immunosuppressive treatment including CsA for at least 12 months [
25,
32,
58]. In contrast, in the two double-blind controlled trials that failed to show efficacy, patients were either not immunosuppressed [
19] or CsA was withdrawn 6 months post-transplantation [
42]. In the latter case, the slope of the grafts effects was reported to have regressed after discontinuation of the immunosuppressive regimen, supporting the idea that a rejection response developed and compromised the long-term benefit of the graft [
42]. In the present study, we found that the striatum of rats overexpressing α-synuclein developed a marked chronic inflammatory process and that this likely constituted a hostile environment for cell engraftment and that its repression using CsA administration promoted the long-term survival VM grafts and especially the dopamine neurons. These data obtained in a novel rat model recapitulating several relevant features of the human condition speak in favor for the use of immunosuppression in clinical trials using allogeneic tissue.
Additional experiments in other α-synuclein models of PD, including a novel mouse model with temporal control of α-synuclein expression, confirmed the unexpected restorative effect of motor and cognitive function induced by CsA.
The therapeutic effect of CsA was first documented 20 years ago in the 6-OHDA- and MPTP-induced models of PD using a neuroprotective design [
38,
40,
46,
49]. More recently, genetic manipulation and pharmacological inhibition of calcineurin using the immunosuppressant FK506 was shown to modulate α-synuclein toxicity and to afford neuroprotection in in vitro and in vivo models of PD [
9,
20,
36,
56]. However, the cell-autonomous mechanisms mediating neuronal survival have not been described and we now show that CsA triggers autophagy and by so doing promotes the clearance of α-synuclein both in cultured neurons and in vivo.
Our experiments in the MPTP model reveal that CsA affects other relevant mechanisms such as the activation of DARPP-32 in striatal medium-size spiny neurons. DARPP-32 integrates signals from the nigral dopaminergic neurons and from the cortical glutamatergic projections [
54]. While no restoration of the dopaminergic pathway was observed in the MPTP model, we reported a decrease in the striatal expression of GLT-1 back to the level of the control group following CsA treatment, reflecting a reduction in the glutamatergic tone, which could have resulted in increased activation of DARPP-32. In addition, DARRP-32 is a known target of calcineurin and CsA treatment stimulates the function of DARPP-32 by modulating its phosphorylation status [
26,
41]. Notably in this experiment, mice were euthanized 48 h after the last injection of CsA to allow for washout of the drug which may explain why only the chronic effect of CsA was found in this experiments while no significant acute effect of CsA treatment within groups was observed. These mechanisms may, at least partly, contribute to the functional recovery seen in this model and possibly also in the α-synuclein-based models. CsA induced additional mechanisms such as axonal sprouting as shown by the increased striatal expression of SCG-10. Steiner and colleagues have previously reported the neurotrophic action of immunosuppressive drugs [
52]; however, the absence of TH recovery in the MPTP model suggests that alternate neuronal sub-types may undergo axonal re-modeling/sprouting.
Although both CsA and FK506 repress calcineurin phosphatase activity, this indirect inhibition is mediated via their interaction with cyclophilin and FKBP12, respectively. Thus, it cannot be inferred that FK506 would exert a therapeutic effect via the same pathways as described here for CsA.
No PD patients, including subjects who received a sham or imitation surgery in the cell therapy trials, have been maintained under CsA treatment or other immunosuppressive treatment long enough to determine its impact on the course of the disease. Nevertheless, in a report from the open-label Swedish transplantation trial, Hagell and colleagues (1999) described the clinical progression before and after sequential bilateral grafting in PD patients who were under immunosuppression. Interestingly, patients with unilateral transplants showed a bilateral improvement in motor function even before the second transplantation was performed, while in receipt of immunosuppressive therapy including CsA [
22]. Considering our novel findings, it cannot be ruled out that the immunosuppressive treatment received by these patients, which included CsA, contributed to the clinical amelioration by triggering mechanisms similar to those observed in our pre-clinical models. This hypothesis can only be verified by a proper assessment of the therapeutic effect of CsA in PD patients.
Acknowledgements
The study was supported by grants from the Fondazione Telethon, the Huffington Foundation, the Swedish Research Council, the Lund University Multipark program, Parkinsonfonden and the U.S Department of Veterans Affairs Merit Review Program. The authors thank Eduardo Nusco, Donatella Montanaro, Jenny Johansson, Elsy Ling, Bengt Mattsson and Michael Sparrenius for excellent technical assistance, Dr. Roger Barker for stimulating discussions in the preparation of the manuscript.
Competing interest
The authors declare that they have no conflict of interest.
Authors’ contributions
AT, MJC, AB, CKM and MD designed the experiments, WOW, DW, YC-S, RI and MD performed the experiments in rats.AT, YC-S, RI, NJH and MD performed experiments in the AAV-based model and the Thy1-alpha-synuclein model. MJC, MDS and CKM performed experiments in the MPTP model. AT, SB and RI performed the in vitro study. AT, MJC, YC-S and MD analyzed the data. AT, MJC, NJH, AB, CKM and MD drafted the manuscript. All authors read and approved the final manuscript.