Direct α-synuclein promoter transactivation by the tumor suppressor p53

Background Parkinson’s disease (PD) is a motor disease associated with the degeneration of dopaminergic neurons of the substantia nigra pars compacta. p53 is a major neuronal pro-apoptotic factor that is at the center of gravity of multiple physiological and pathological cascades, some of which implying several key PD-linked proteins. Since p53 is up-regulated in PD-affected brain, we have examined its ability to regulate the transcription of α-synuclein, a key protein that accumulates in PD-related Lewy bodies. Results We show that pharmacological and genetic up-regulation of p53 expression lead to a strong increase of α-synuclein protein, promoter activity and mRNA levels. Several lines of evidence indicate that this transcriptional control is due to the DNA-binding properties of p53. Firstly, p53 DNA-binding dead mutations abolish p53 regulation of α-synuclein. Secondly, the deletion of p53 responsive element from α-synuclein promoter abrogates p53-mediated α-synuclein regulation. Thirdly, gel shift and chromatin immunoprecipitation studies indicate that p53 interacts physically with α-synuclein promoter both in vitro and in a physiological context. Furthermore, we show that the depletion of endogenous p53 in cells as well as in knockout mice down-regulates α-synuclein transcription. Conclusions Overall, we have identified α-synuclein as a new transcriptional target of p53 and delineated a cellular mechanism feeding the accumulation of toxic aggregated α-synuclein in PD. This original α-syn regulatory mechanism may be central to PD-related cell death and may lead to novel opportunities to design alternative neuroprotective strategies in PD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0079-2) contains supplementary material, which is available to authorized users.


Background
Parkinson's disease (PD) is a major age-related motor disease in which several causative genes have been identified. Amongst these genes, α-synuclein (α-syn) has caught a special attention given its important role in PD etiology [1,2]. Thus, α-syn is a small phosphoprotein, that accumulates in intracellular inclusions named Lewy bodies in most of sporadic and genetic PD cases [3]. We previously showed that it exerts an anti-apoptotic phenotype by down-regulating p53 expression and transcriptional activity in neuronal cells [4,5]. Importantly this neuroprotective phenotype can be abrogated by various processes leading to its accumulation and/or aggregation.
Thus, we have shown that 6-hydroxydopamine, an endogenously produced dopamine catabolite frequently used to trigger PD ex-vivo and in vivo, leads to α-syn aggregation and consequently, abolishes α-syn ability to repress p53 [6].
It is interesting to note that several proteins such as DJ-1 and parkin, which when mutated trigger familial forms of PD, have been shown to regulate and, to be regulated by p53 [7][8][9][10][11]. In this context, the ability of αsyn to regulate p53 led us to postulate that p53 could also control α-syn levels as part of a feedback process driving their cellular homeostasis in neurons. Given the canonical transcription factor properties of p53, we have examined the putative p53-dependent control of α-syn transcription in a pathophysiological context. Our study demonstrates that both selective pharmacological treatments and genetic manipulation of p53 by overexpression or invalidation approaches trigger a modulation of α-syn transcription by a mechanism implying the physical interaction of p53 with α-syn promoter. Thus, the deletion of putative p53 responsive element of α-syn mouse promoter leads to full abolishment of p53-mediated α-syn transcription up-regulation. ChIP (chromatin immunoprecipitation) and gel shift analyses demonstrate that the p53 and α-syn interaction occurs in physiological context in absence of any transcription co-factor. Importantly, analysis of α-syn protein and mRNA levels in p53 knockout mouse brain further documents the transcriptional control of α-syn by p53 in vivo. Overall, our data identify α-syn as a novel p53 transcriptional target and thus, delineate a functional interplay driving their cellular homeostasis. Our study pinpoints to the fact that the disruption of this cellular dialogue linked to the dysfunction of any of these two partners may contribute to PD pathology.

Fig. 3
Mouse and human endogenous p53 control α-syn transcription. Control mouse fibroblasts (MEF, p19 arf-/-, black bars) or MEF devoid of p53 (p19 arf-/-p53 -/-, white bars) were assessed for α-syn protein (a and b), promoter transactivation (c) and mRNA levels (d) in basal conditions as described in the Methods section. α-syn protein (e, f), promoter transactivation (g) and mRNA levels (h) were analyzed in HCT116 control (HCT +/+ , black bars) or p53-deficient (HCT -/-, white bars) cells as described in the Methods section. Bars in A-D represent the means ± SEM of 3-4 independent experiments performed in duplicates or triplicates and are expressed as percentage of control p19 arf-/-(a-d) or HCT +/+ (e-h) cells. Actin expression is provided as a gel loading control in (a, e). Statistical analyses were performed with GraphPad Prism software by using homoscedastic, unpaired Student's t-test. Significant differences are: **p < 0.01, and ***p < 0.001 Additional file 2: Figure S2C) were observed in HAP -/cells.
In order to establish that our ex-vivo observations also stand in vivo, we have compared α-syn protein (left brain hemisphere) and mRNA status (right brain hemisphere) of wild-type mouse harboring (p53 +/+ ) or lacking (p53 -/-) endogenous p53. p53 knockout mouse brains show a similar reduction of α-syn protein expression (-58.9 % ± 11.6, n = 6, Fig. 4a) and mRNA levels (-51.8 % ± 10.1, n = 9, Fig. 4b). Overall, the above-described data demonstrate that pharmacological modulation or genetic manipulation of endogenous p53 control α-syn mRNA transcription and protein levels in various cell types from both murine and human origin as well as in mouse brain.
α-syn is a direct p53 transcriptional target The question arose as to whether p53 could directly or indirectly modulate α-syn promoter transactivation. An in silico bioinformatic search for p53 DNA-binding consensus motif [16] identified a unique motif corresponding to half of the canonical p53 responsive element in the mouse α-syn promoter (-970 → -967, Fig. 6a). We first examined the influence of the deletion of this putative p53 responsive element on p53-mediated modulation of α-syn transcription. First, as expected, we confirmed that, conversely to the phenotype observed after depletion of endogenous p53, its overexpression (see Fig. 6b, upper panel) increases α-syn wild-type full length α-syn mouse promoter transactivation (+123.1 % ± 16.6, n = 12, Fig. 6b compare black bars). This p53related phenotype is completely abolished by the removal of the p53-binding domain on the α-syn promoter construct (Δ α-syn prom., Fig. 6b, compare white bars). Two lines of additional data definitely flag-up α-syn as a transcriptional target of p53. Firstly, we performed electrophoretic mobility shift assay (EMSA) where recombinant wild-type p53 protein and biotinylated α-synderived probe encompassing its p53 DNA-binding motif were incubated together. This bi-molecular reaction allows monitoring a genuine physical interaction between the two partners in absence of any cellular modulator or intermediate. Figure 6c illustrates the gel shift elicited by p53/α-syn probe physical interaction (Fig. 6c, compare lanes 1 and 2). The specificity of this interaction was demonstrated by its blockade by pre-incubation of p53 recombinant protein with two distinct p53 antibodies pab421/DO1 (anti-p53, lane 3 and 6 respectively) or by incubation with an excess of specific non-biotinylated αsyn probes (comp-sp, lanes 4 and 5). Secondly, we showed by ChIP experiments that endogenous p53 immunoprecipitation yielded a α-syn promoter PCRamplified fragment containing the p53 DNA-binding motif (Fig. 6d, compare control lane 2 with IPed lane 3). This set of deletion-based analysis, gel shift data and ChIP experiments all support our in silico prediction and demonstrate that α-synuclein behaves as a genuine direct p53 transcriptional target.

Discussion
p53 is a transcription factor, which main physiological function is to regulate genes involved in the control of cell cycle, DNA repair and apoptosis [17,18]. Thus, p53associated gene modulation normally stops cell growth Fig. 4 p53 control α-syn transcription in vivo. Analyses of α-syn protein (a) and mRNA levels (b) in control (p53 +/+ , black bars) or TP53 gene invalidated (p53 -/-, white bars) mouse brain as described in the Methods section. Bars represent the means ± SEM of n = 6 (a) or n = 9 animals (b) and are expressed as percentage of control p53 +/+ mouse brain samples. Actin expression (a) is provided as protein loading control. Statistical analyses were performed with GraphPad Prism software by using homoscedastic, unpaired Student's t-test. Significant differences are: **p < 0.01, and ***p < 0.001 by blocking the cell cycle at the G1/S phase and triggers a pro-apoptotic cascade [18]. Moreover, p53 is a proapoptotic protein strongly linked to several neurodegenerative disorders, amongst which Parkinson's disease [19]. Thus, post-mortem studies of PD-affected patients have shown abnormal p53 expression and the evaluation of the impact of toxin-induced PD demonstrated a strong contribution of p53 in dopaminergic cell death. Finally, several PD-causative gene products including DJ-1 and parkin, have been shown to Statistical analyses were performed with GraphPad Prism software by using One-way ANOVA analysis of variance coupled to a Newman Keuls post-hoc test. Significant differences are: **p < 0.01, and ***p < 0.001 Fig. 6 p53 interacts physically with murine α-syn promoter. a Scheme of the region of wild-type α-synuclein promoter (WT α-syn prom) with the p53 DNA-binding motif indicated in bold. The -970-967 (5'-CATG-3') region underlined has been deleted (Δα-syn prom). b SH-SY5Y cells were co-transfected with either empty pcDNA3 vector (EV, lanes -) or p53 cDNA (p53, lanes +) and WT α-syn prom (black bars) or Δα-syn prom (empty bars) constructs then promoter activities were measured as described in the Methods section. p53 expression was controlled by anti-Flag antibodies as described in Methods. Bars represent the means ± SEM of 4 independent experiments performed in triplicates and are expressed as percentage of control EV-transfected cells. c EMSA analysis of the interaction of wild-type recombinant p53 (p53r) with α-syn-derived biotinylated probe encompassing the consensus sequence shown in (a). Reactions were carried out in absence (-) or in the presence (+) of either an excess of specific cold probes (comp-sp, see lanes 4, 5) or p53-directed antibodies (anti-p53 pab421/DO1, lanes 3, 6) and analyzed as described in the Methods. Free probe control is shown in lane 1. d ChIP analysis of the interaction between endogenous p53 with α-syn endogenous promoter in MEF cells by endpoint semi-quantitative PCR (upper gel) or real time PCR (histogram) as described in Methods. In (d), lanes 1-5 correspond to 100 bp DNA ladder (STD), normal mouse IgG ChIP (CT), p53 ChIP (IP), input (INP) and no template control (H 2 O), respectively. Statistical analysis was performed with GraphPad Prism software by using either One-way ANOVA analysis of variance coupled to a Newman Keuls post-hoc test (b) or homoscedastic, unpaired Student's t-test (d). Significant differences are: *p < 0.05, ***p < 0.001, and ns for non-significant control and to be controlled by p53 ex-vivo and in vivo [19,20].
Our study unravels for the first time α-syn as an additional transcriptional target of p53. This conclusion stands on five independent lines of data. Firstly, overexpression of p53 or its pharmacological activation enhance α-syn promoter activity, mRNA and protein levels in human and murine cells from various origins while p53 gene depletion triggers the opposite phenotype in cells as well as in vivo, in mouse brain; secondly, DNAbinding dead mutations of p53 abolish p53-induced α-syn transactivation both ex-vivo; thirdly, the deletion of a putative p53 DNA-binding element on the α-syn promoter prevents p53-mediated increase of α-syn promoter activation; fourth, EMSA experiments indicated that recombinant p53 and α-syn promoter-derived probes physically interact directly without the need of any cofactor; fifthly, ChIP analysis demonstrates the physical association of endogenous p53 with α-syn promoter in an ex-vivo physiological context.
Very few works aimed at studying the transcriptional regulation of α-syn have been performed. Importantly, previous data indicated that α-syn mRNA levels could be modulated by PD inducers like MPTP (1-Methyl-4phenyl-1,2,3,6-tetrahydropyridine hydrochloride) [21] and appeared increased in PD-affected brains [22]. Thus, our data allowed the establishment of a molecular mechanism that ultimately may contribute to the transcriptional modulation of α-syn in sporadic PD.
We previously established that α-syn lowers both p53 protein levels and transcriptional activity in neuronal cell lines [5] and that this control was abolished by 6-hydroxydopamine that triggers both α-syn oxidation and aggregation and proteasomal inhibition [6]. This study, together with our present data, unravels a functional interplay between α-syn and p53 that could be disrupted in pathological conditions. Thus, in normal conditions, α-syn represses p53 [1,5]. This reduction of p53 triggers lowering of α-synuclein transactivation and protein levels (present study), which in turn restores p53 physiological levels. We speculate that this reciprocal physiological control, which allows the homeostasis of both proteins, may be impaired in PD due to dysfunctions of either α-syn or p53. Thus, in those cases where the function of α-syn is hampered by its aggregation or by pathogenic mutations, one can anticipate the accumulation of p53 and by consequence the establishment of a deleterious p53-mediated transcriptional feeding of α-syn increase. Alternatively, since p53 is at the crossroad of multiple signaling cascades involved in PD pathogenesis, one can suppose that its abnormal activation could lead to excessive α-syn transcription and production. Since α-syn aggregation is strongly linked to its protein levels, one can envision that p53 could contribute to α-syn aggregation and toxicity via exacerbation of its transcriptional regulation.

Conclusions
Our work demonstrates that α-syn promoter harbors a functional p53 responsive element. Thus, p53 interacts with the mouse α-syn promoter and up-regulates α-syn transcription. Pharmacological and genetic manipulation of p53 impacts α-syn regulation and importantly, endogenous p53 regulates α-syn transcription ex-vivo and in vivo. This first delineation of α-syn as a physiological transcriptional target of p53 unraveling a functional dialogue between these two proteins allows proposing that, in a PD-linked pathological context, α-syn toxicity could be likely the consequence of a loss of its physiological interplay with p53.

Pharmacological and UV irradiation-mediated modulation of p53
Activation of p53 was achieved by UV-light irradiation of the cells by means of a cross-linker apparatus (254 nm bulb). Various cell types were cultivated in 35 mm dishes and submitted to a double optimal crosslink treatment (60 sec, 120 mJ/cm 2 ) when they reached 80 % confluence then left 24 hours at 37°C in a 5 % CO 2 atmosphere. The pharmacological modulation of p53 was obtained after 12 hours incubations with etoposide (150 μM) or leptomycin (10 nM). After treatments, cells were recovered and protein and RNA analyses were monitored as described below.

Plasmid constructs and transfections approaches
The generation of wild-type and R273H flag-tagged p53 mutant of human p53 flag-tagged coding sequences in the mammalian expression vector pcDNA3.1 (+) has been extensively described [24]. To generate the R248W p53 mutant, we used the site-directed mutagenesis kit from Stratagene along with the forward p53R248WS

Luciferase-based reporter assays
The transactivation of the wild-type and mutated SNCA mouse promoter described in the plasmid constructs section was followed by recording the luciferase reporter gene activity 24 hours after co-transfection of 0,5-1 μg of the above cDNAs and 0,2-0,5 μg of β-galactosidase cDNA (in order to normalize for transfection efficiencies) by means of lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions (Invitrogen). When necessary, in a subset of experiments, 0,5-1 μg of empty pcDNA3.1, wild type or mutated p53 were cotransfected.

RNA extraction, reverse transcription and real-time PCR analysis
RNA from cells and RNA later (Qiagen) stabilized mouse brains (right hemisphere) were extracted and treated with DNAse using RNeasy or RNeasy Plus Universal Mini kits respectively following manufacturer's instructions (Qiagen). Two μg of total RNA were reverse transcribed (GoScript Reverse Transcriptase, Promega) using Oligo-dT priming. Then, samples were subjected to real-time PCR by means of a Rotor-Gene 6000 apparatus (Qiagen), using the SYBR Green detection protocol. Gene-specific primers were designed with the Universal Probe Library Assay Design Center software (Roche Applied Science). Expression levels of mouse (forward: 5′-GGG-CTT-TGA-CAG-GAT-GGA-AGG-GCA-TGT-CTC-CAG-CGA-AAG-G-3′) encompassing the putative p53 responsive element. Protein-probe complexes were then resolved by electrophoresis on a 5 % native polyacrylamide gel at 4°C, transferred to a positively charged nylon membrane (Thermo Scientific), cross-linked (UV-light crosslinker equipped with a 254 nm bulb) and revealed by means of streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. When indicated, in order to confirm specific DNA-protein interaction, we performed a 15-30 min pre-incubation at 4°C with either an excess (4 pmol) of unlabeled specific and non-related control DNA or pab421 before adding the biotin-labeled probes.

Chromatin Immunoprecipitation assay (ChIP)
ChIP assay was performed according to EZ-ChIP kit instructions (Millipore). Briefly, 10 7 cells were fixed with formaldehyde (1 % final concentration), treated with glycine to quench unreacted formaldehyde and recovered in cold phosphate buffered saline (PBS) containing the protease inhibitor cocktail II. Pelleted cells were lysed in the SDS lysing buffer and sonicated on ice in order to obtain chromatin fragments of about 200-500 bp in size. After a preclearing step using protein G Agarose, immunoprecipitation was performed either with anti-p53 primary antibody (pab421, Enzo Life Sciences) or normal mouse IgG as a negative control. Immunocomplexes were then incubated with a solution of protein Gagarose. After elution of the immunocomplexes from beads, crosslinks were reversed and RNA and protein eliminated by RNAse and proteinase K treatments. DNA was purified and subjected to a standard end-point and real-time PCR using primers (forward: 5′-CGC-CTA-GAG-AAG-ACC-AAC-TAC-AGC-TGC-3′; reverse: 5′-GCA-CTA-AGC-TTC-CAC-CAT-CCA-GCA-CTC-AAC-3′) specific for the -1061/-852 DNA region (210 bases-long amplicon) upstream the start codon of mouse SNCA gene. We calculated the enrichment as the ratio of the amplification efficiency of the ChIP sample over that of the IgG.

Statistical analysis
Statistical analyses were performed with GraphPad Prism software (www.graphpad.com version 4.00 for Windows, San Diego, California USA). All groups of samples analyzed by Student's t-test have passed a normality test to assure Gaussian distribution of values and precision concerning the type of test (unpaired versus paired and homoscedastic versus heteroscedastic) are provides in figures legends. Analysis of more than two groups of variables (normality test passed) was performed by One-way ANOVA with Newman-Keuls's post-hoc test. Significant differences are: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns = non significant.