Study of the transcriptional regulation of α-synuclein

α-synuclein is a small presynaptic neuronal protein, encoded by the SNCA gene, that is implicated genetically and neuropathologically in Parkinson’s disease (PD). A large body of evidence has established that PD pathogenesis is closely linked to increased levels of SNCA; however to date, the biochemical pathways and transcriptional elements that control SNCA expression are still obscure. Previous experiments in our laboratory in the PC12 cell line demonstrated that the transcription factor ZSCAN21 binds to the intron 1 region of the Snca gene and is strongly involved in its transcriptional regulation. Therefore, in the current experiments, we wished to characterize further the role of ZSCAN21 in Snca transcriptional regulation in primary cultures and in vivo. We find that in vivo ZSCAN21 is expressed in neurons and its levels are developmentally regulated in different brain regions where ASYN is also detected. Further, we confirmed through Chromatin Immunoprecipitation its presence in a binding complex in the intron 1 region of the Snca gene in rat cortical neuronal cultures. Importantly, lentiviral-mediated silencing of Zscan21 increased significantly the promoter activity of Snca as well as its mRNA and protein levels in such cultures. In contrast, Zscan21 mediated silencing in differentiated neurosphere cultures reduced Snca levels. Stereotaxic delivery of adeno-associated virus against Zscan21 in the postnatal and adult hippocampus, an area linked with the non-motor symptoms of PD, revealed no significant alterations in Snca levels, despite efficient Zscan21 knockdown. Interestingly, Zscan21 overexpression in cortical neurons with adenoviruses led to robust mRNA but negligible protein expression, suggesting that ZSCAN21 protein levels are tightly regulated post-transcriptionally and / or post-translationally. Therefore, overall, our study demonstrates that ZSCAN21, a transcription factor whose levels are under strict posttranscriptional / posttranslational control in neurons, is diversely implicated in the transcriptional regulation of Snca in respect to the developmental stage, at least in in vitro primary neuronal settings. In vivo, however, the unaltered Snca levels observed following Zscan21 downregulation, imply the presence of alternative or perhaps compensatory mechanisms that regulate Snca transcription in such settings. Furthermore, in a genetic case control study of the ZSCAN21 binding site in SNCA intron 1, we did not find polymorphisms between PD patients and controls, suggesting that genetic diversity within this region does not contribute to disease pathogenesis. Overall, given the diverse effects in cell culture, and the lack of discernible in vivo effects, and although further studies are needed, our work does not provide sufficient support for the idea of targeting ZSCAN21 in order to manipulate SNCA levels in synucleinopathy models

Μελέτη της μεταγραφικής ρύθμισης της α-συνουκλεΐνης

α-synuclein is a small presynaptic neuronal protein, encoded by the SNCA gene, that is implicated genetically and neuropathologically in Parkinson’s disease (PD). A large body of evidence has established that PD pathogenesis is closely linked to increased levels of SNCA; however to date, the biochemical pathways and transcriptional elements that control SNCA expression are still obscure. Previous experiments in our laboratory in the PC12 cell line demonstrated that the transcription factor ZSCAN21 binds to the intron 1 region of the Snca gene and is strongly involved in its transcriptional regulation. Therefore, in the current experiments, we wished to characterize further the role of ZSCAN21 in Snca transcriptional regulation in primary cultures and in vivo. We find that in vivo ZSCAN21 is expressed in neurons and its levels are developmentally regulated in different brain regions where ASYN is also detected. Further, we confirmed through Chromatin Immunoprecipitation its presence in a binding complex in the intron 1 region of the Snca gene in rat cortical neuronal cultures. Importantly, lentiviral-mediated silencing of Zscan21 increased significantly the promoter activity of Snca as well as its mRNA and protein levels in such cultures. In contrast, Zscan21 mediated silencing in differentiated neurosphere cultures reduced Snca levels. Stereotaxic delivery of adeno-associated virus against Zscan21 in the postnatal and adult hippocampus, an area linked with the non-motor symptoms of PD, revealed no significant alterations in Snca levels, despite efficient Zscan21 knock-down. Interestingly, Zscan21 overexpression in cortical neurons with adenoviruses led to robust mRNA but negligible protein expression, suggesting that ZSCAN21 protein levels are tightly regulated post-transcriptionally and / or post-translationally. Therefore, overall, our study demonstrates that ZSCAN21, a transcription factor whose levels are under strict posttranscriptional / posttranslational control in neurons, is diversely implicated in the transcriptional regulation of Snca in respect to the developmental stage, at least in in vitro primary neuronal settings. In vivo, however, the unaltered Snca levels observed following Zscan21 downregulation, imply the presence of alternative or perhaps compensatory mechanisms that regulate Snca transcription in such settings. Furthermore, in a genetic case control study of the ZSCAN21 binding site in SNCA intron 1, we did not find polymorphisms between PD patients and controls, suggesting that genetic diversity within this region does not contribute to disease pathogenesis. Overall, given the diverse effects in cell culture, and the lack of discernible in vivo effects, and although further studies are needed, our work does not provide sufficient support for the idea of targeting ZSCAN21 in order to manipulate SNCA levels in synucleinopathy models.Η νόσος του Πάρκινσον (ΝΠ) αποτελεί μια χρόνια νευροεκφυλιστική νόσο με άγνωστη μέχρι σήμερα αιτιολογική θεραπεία. Ένα από τα σημαντικότερα γονίδια που εμπλέκονται στη παθολογία της ΝΠ είναι η α-συνουκλεΐνη (SNCA), η οποία κωδικοποιεί για μια μικρή, νευρωνική, προσυναπτική πρωτεΐνη. Πλήθος μελετών υποστηρίζουν ότι η υπερέκφραση της SNCA οδηγεί σε φαινότυπους που προσομοιάζουν με τη ΝΠ σε διάφορα κυτταρικά και ζωικά μοντέλα. Παρόλα αυτά τα βιοχημικά μονοπάτια καθώς και τα ρυθμιστικά στοιχεία που ελέγχουν τα επίπεδα έκφρασής της παραμένουν σε μεγάλο βαθμό αδιευκρίνιστα. Προηγούμενα πειράματα στο εργαστήριο μας, στην κυτταρική σειρά PC12, έχουν δείξει ότι ο μεταγραφικός παράγοντας ZSCAN21 προσδένεται στη ρυθμιστική περιοχή του πρώτου εσωνίου της Snca και συμμετέχει ενεργά στη μεταγραφική της ρύθμιση. Βασιζόμενοι σε αυτό το εύρημα, στη παρούσα μελέτη διερευνήσαμε περαιτέρω τη δράση του ZSCAN21 στη ρύθμιση των επιπέδων της Snca σε πρωτογενείς καλλιέργειες νευρώνων καθώς και σε εγκεφάλους από επίμυες in vivo. Διαπιστώσαμε τη νευρωνική έκφραση του ZSCAN21 καθώς και τη διαφορική του ρύθμιση σε διακριτά αναπτυξιακά στάδια σε περιοχές του εγκεφάλου (από επίμυ) όπου εντοπίζεται και η ASYN. Επιπλέον, μέσω πειραμάτων ανοσοκατακρήμνισης χρωματίνης (CHIP), επιβεβαιώσαμε τη συμμετοχή του ZSCAN21 στο ρυθμιστικό σύμπλοκο-πρόσδεσης στη περιοχή του πρώτου εσωνίου της Snca σε καλλιέργειες φλοιού από επίμυ. Στοχευμένη σίγαση της έκφρασης του Zscan21 σε πρωτογενείς καλλιέργειες νευρώνων μέσω λεντι-ιών οδήγησε σε σημαντική αύξηση της Snca τόσο σε επίπεδο ενεργότητας του υποκινητή της όσο και σε επίπεδο mRNA και πρωτεΐνης. Αντίθετα, σίγαση του Zscan21 σε καλλιέργειες νευρικών βλαστικών κυττάρων (νευροσφαιρών, neurosphere cultures) είχε σαν αποτέλεσμα τη μείωση των επιπέδων της Snca. Στερεοταξική έγχυση αδενοσχετιζόμενων ιών (AAVs) έναντι του Zscan21 σε μετεμβρυϊκούς (postnatal) και ενήλικες επίμυες στην περιοχή του ιππόκαμπου, η οποία συνδέεται κύρια με τα μη κινητικά συμπτώματα της ΝΠ, δε μετέβαλε σημαντικά τα επίπεδα της Snca. Πειράματα υπερέκφρασης του Zscan21 σε πρωτογενείς φλοιϊκές καλλιέργειες από επίμυ μέσω αδενο-ιών (AVs) δεν αύξησαν σημαντικά τα επίπεδα της πρωτεΐνης του ZSCAN21, παρά τη σημαντική αύξηση στο επίπεδο του mRNA του Zscan21, υποδηλώνοντας ότι τα επίπεδα του ZSCAN21 πρέπει να υπόκεινται σε αυστηρό μετα-μεταγραφικό ή / και μετα-μεταφραστικό έλεγχο. Λαμβάνοντας υπόψιν τα παραπάνω αποτελέσματα διαπιστώνεται ότι ο ZSCAN21, ένας μεταγραφικός παράγοντας του οποίου τα πρωτεϊνικά επίπεδα ρυθμίζονται αυστηρά, εμπλέκεται ποικιλοτρόπως στη μεταγραφική ρύθμιση της Snca ανάλογα με το αναπτυξιακό στάδιο, τουλάχιστον στα in vitro συστήματα πρωτογενών καλλιεργειών που μελετήσαμε. Παρόλα αυτά, το γεγονός ότι δε παρατηρήθηκαν σημαντικές αλλαγές στα επίπεδα της Snca έπειτα από σίγαση του Zscan21 σε επίμυες ιn vivo, υποδηλώνει τη παρουσία αντιρροπιστικών μηχανισμών που συμμετέχουν στη ρύθμιση της. Επιπλέον, ανάλυση δειγμάτων ασθενών με ΝΠ και ατόμων ελέγχου, σχετικά με την εύρεση πιθανών πολυμορφισμών τόσο στη θέση πρόσδεσης του ZSCAN21 όσο και στο ευρύτερο τμήμα στη περιοχής του πρώτου εσωνίου της SNCA που περιέχει επιπλέον ρυθμιστικά στοιχεία δεν αποκάλυψε σημαντικές διαφορές μεταξύ των δύο ομάδων. Το εύρημα αυτό υποδηλώνει ότι η γενετική πολυμορφία μέσα στη συγκεκριμένη περιοχή δε φαίνεται να συνεισφέρει στη παθογένεια της νόσου. Συνολικά, η πολυπλοκότητα της δράσης του ZSCAN21 στη ρύθμιση της Snca σε διαφορετικά συστήματα πρωτογενών καλλιεργειών νευρώνων, σε συνδυασμό με την απουσία επίδρασης του ZSCAN21 στα επίπεδα της SNCA στα in vivo πειράματα, συνηγορούν στο ότι η στόχευση του ZSCAN21 ως πιθανό θεραπευτικό μέσο δε φαίνεται να αποτελεί τη κατάλληλη επιλογή για τη ρύθμιση των επιπέδων της SNCA

Functional dissection of the alpha-synuclein promoter: transcriptional regulation by ZSCAN21 and ZNF219

Alpha-Synuclein (SNCA) is an abundant neuronal protein involved in synaptic neurotransmission. SNCA expression levels have been strongly implicated in Parkinson’s disease pathogenesis. We have previously demonstrated that in the PC12 cell line elements in intron 1 may mediate SNCA transcriptional regulation in response to neurotrophins. We have now identified transcription factor (TF) binding sites in intron 1 and the 5’-promoter of SNCA. A binding site for the TF zinc finger and SCAN domain containing (ZSCAN) 21 in the 5’-region of intron 1 is required for intron 1 transcriptional activity. Small interfering RNA against ZSCAN21 inhibits activation in the luciferase assay and diminishes SNCA protein levels in naive and neurotrophin-treated PC12 cells and in primary cultured cortical neurons, demonstrating that ZSCAN21 is a novel transcriptional regulator of SNCA in neuronal cells. The 5’-promoter of SNCA has a complex architecture, including multiple binding sites for the TF zinc finger protein (ZNF) 219, which functions as both an activator and a repressor. Targeting ZSCAN21 or other TFs controlling SNCA transcriptional activity may provide novel therapeutic avenues not only for Parkinson’s disease but also for other synucleopathies

Complex effects of the ZSCAN21 Transcription factor on transcriptional regulation of α-synuclein in primary neuronal cultures and in Vivo

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is strongly implicated in Parkinson disease (PD). PD pathogenesis is linked to increased SNCA levels; however, the transcriptional elements that control SNCA expression are still elusive. Previous experiments in PC12 cells demonstrated that the transcription factor zinc finger and SCAN domain containing 21 (ZSCAN21) plays an important regulatory role in SNCA transcription. Currently, we characterized the role of ZSCAN21 in SNCA transcription in primary neuronal cultures and in vivo. We found that ZSCAN21 is developmentally expressed in neurons in different rat brain regions. We confirmed its binding in the intron 1 region of SNCA in rat cortical cultures. Lentivirus mediated silencing of ZSCAN21 increased significantly SNCA promoter activity, mRNA, and protein levels in such cultures. In contrast, ZSCAN21 silencing reduced SNCA in neurosphere cultures. Interestingly, ZSCAN21 overexpression in cortical neurons led to robust mRNA but negligible protein expression, suggesting that ZSCAN21 protein levels are tightly regulated post-transcriptionally and/or post-translationally in primary neurons. Efficient adeno-associated virus-mediated knockdown of ZSCAN21 in the postnatal and adult hippocampus, an area linked with non-motor PD symptoms, revealed no significant alterations in SNCA levels. Overall, our study demonstrates that ZSCAN21 is involved in the transcriptional regulation of SNCA in primary neuronal cultures, but the direction of the effect is variable, likely depending on neuronal maturation. However, the unaltered SNCA levels observed following ZSCAN21 down regulation in the rat brain, possibly due to compensatory mechanisms, imply that ZSCAN21 is not a master regulator of SNCA in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc

Correction: Loss of β-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells.

[This corrects the article DOI: 10.1371/journal.pone.0060674.]

Loss of β-glucocerebrosidase activity does not affect alpha-synuclein levels or lysosomal function in neuronal cells.

To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson's disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD

Loss of β-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells

To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson's disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD. © 2013 Dermentzaki et al

Regulation of alpha-synuclein expression in cultured cortical neurons

P>Alpha-synuclein (SNCA) is a predominantly neuronal protein involved in the control of neurotransmitter release. The levels of SNCA expression are closely linked to the pathogenesis of Parkinson’s disease; however, the biochemical pathways and transcriptional elements that control SNCA expression are not well understood. We previously used the model system of neurotrophin-mediated PC12 cell neuronal differentiation to examine these phenomena. Although these studies were informative, they were limited to the use of a cell line; therefore, in the current work, we have turned our attention to cultured primary rat cortical neurons. In these cultures, SNCA expression increased with time in culture, as the neurons mature. Luciferase assays based on transient transfections of fusion constructs encoding components of the transcriptional control region of SNCA identified various promoter areas that have a positive or negative effect on SNCA transcription. Intron 1, previously identified by us as an important regulatory region in the PC12 cell model, cooperates with regions 5’ to exon 1 to mediate gene transcription. Using selective pharmacological tools, we find that tyrosine kinase receptors and the phosphatidyl-inositol 3 kinase signaling pathway are involved in mediating these effects. The exogenous application of the neurotrophin brain-derived neurotrophic factor (BDNF) is sufficient on its own to promote the transcriptional activation of SNCA through this pathway, but a neutralizing antibody against BDNF failed to affect SNCA transcription in maturing cultures, suggesting that BDNF is not the main factor involved in maturation-induced SNCA transcription in this model. Further in vivo studies are needed to establish the role of neurotrophin signaling in the control of SNCA transcription