Sgt1 Regulates α-Synuclein Subcellular Localization and Expression of Parkinson’s Disease Related Genes, PINK1 and PARK9

The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD). In the present work we have found that SGT1 protected cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. To gain more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in HEK293 cells with an altered SGT1 level. By immunofluorescent staining we have found that in HEK293 cells overexpressing SGT1 α-synuclein was mainly localized in the cytoplasm while in control cells it was present in the nucleus. Accordingly, when SGT1 expression was silenced, α-synuclein was predominantly present in the nucleus. These results were then confirmed by subcellular fractionation and Western blot analysis. Moreover, we have found that altered level of SGT1 in HEK293 cells influenced the expression of PD related genes, PINK1 and PARK9. Altogether, our results point to SGT1 as an important factor that might be involved in the pathogenesis of Parkinson’s disease (PD).


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the presence of characteristic intracellular inclusions, called Lewy bodies (LBs), composed of misfolded α-synuclein, and by selective loss of dopaminergic neurons in substantia nigra. The majority of PD cases are sporadic in origin. However, mutations in several proteins, including α-synuclein, PTEN-induced kinase 1 (PINK1) or Polyamine-Transporting ATPase 13A2 (encoded by PARK9 gene), have been linked to PD [1].
Previous studies have shown that under physiological conditions α-synuclein is involved in regulating membrane lipid content, vesicle trafficking, dopamine metabolism or SNARE-dependent complex assembly [2]. α-Synuclein is localized at presynaptic terminals, however, it is also found in the cytoplasm, mitochondria, endoplasmic reticulum and Golgi apparatus/lysosomes [3]. Nuclear localization of α-synuclein has been described in transgenic Drosophila [4], mice [5] and in cultured cells [6], but its physiological significance is not fully understood. Previously, it has been shown that nuclear α-synuclein promotes neurotoxicity by inhibiting histone acetylation, while sequestration of α-synuclein in the cytoplasm is protective [4]. Such effect can be due to the involvement of α-synuclein in regulation of transcription through binding to DNA [7], histones [8] and chromatin [9]. For instance, it has been shown that α-synuclein is involved in downregulation of Nurr1, a nuclear receptor essential for the development and specification of midbrain dopamine neurons [10]. Binding of α-synuclein to DNA and subsequent down-regulation of multiple cell cycle-related genes has been recently determined by chromatin immunoprecipitation assay (ChIP) followed by next-generation sequencing (ChIP-seq) in Lund human mesencephalic (LUHMES) cells and in transgenic mice overexpressing α-synuclein [7]. SGT1 (suppressor of G2 allele of Skp1) is a component of the CBF3 kinetochore and SCF ubiquitin ligase complexes [11]. Importantly, SGT1 binds Hsp90 and is a component of the chaperone complexes [12]. It can also play a role in innate immunity through stabilization of the NOD1 receptor [13]. SGT1 is highly expressed in neurons of the hippocampus and cortex and in Purkinje and glial cells of the white matter of the cerebellum [14]. Interestingly, SGT1 seems to be involved in several neurodegenerative disorders including Alzheimer's disease (AD) [14], PD or dementia with Lewy bodies (DLB) [15]. In particular, in cortical regions of AD brain, the density of SGT1-stained neurons was lower compared to corresponding regions of control brain [14]. This finding implies that SGT1immunopositive cells may be selectively affected in AD. In turn, SGT1 mRNA level was found to be higher in frontal and temporal cortex of PD and in substantia nigra of DLB brains. Interestingly, SGT1 was not found in Lewy bodies, which are composed mainly of aggregated/phosphorylated α-synuclein [15].
One of the cellular mechanisms that control α-synuclein folding involves chaperone proteins/heat shock proteins (HSPs). Numerous studies have shown that some chaperones and co-chaperones protect cells from α-synuclein aggregation and toxicity [16]. For instance, a novel chaperone/co-chaperone, CacyBP/SIP, inhibits α-synuclein aggregation in in vitro assays and protects cells from toxicity induced by rotenone, an agent that evokes behavioral and histopathological symptoms of PD [17]. Taking into account similar domain organization and high amino acid sequence homology between CacyBP/SIP and SGT1 [18], we hypothesized that SGT1 might also exert beneficial effects counteracting α-synuclein toxicity.
Results obtained in the present work have shown that SGT1 protects HEK293 cells from rotenone-induced toxicity and that it may be involved in regulating α-synuclein partitioning between nucleus and cytoplasm. Moreover, the results point that altered SGT1 level has an effect on the expression of PD-related genes such as PINK1 and PARK9.

Plasmids
List of plasmids used in this work is shown in Table 1. p3×FLAG-CMV-10-SGT1B was prepared as follows: a DNA fragment containing the coding sequence of human SGT1B was amplified by PCR using pDEST17 as a template plasmid (kindly provided by K. Kitagawa, Department of Molecular Medicine, Greehey Children's Cancer Research Institute, San Antonio, TX, USA) and the following primers: forward 5 -TAATAAAGCTTATGGCGGCGGCTGCAGCA-3 and reverse 5 -ACGCGCGAATTCTTAGTACTTTTTCCATCC-3 . The PCR product was digested with HindIII and EcoRI restriction enzymes (both from Thermo Fisher Scientific, Waltham, MA, USA) and introduced into the p3×FLAG-CMV-10 plasmid (Sigma-Aldrich, St. Louis, MO, USA) previously digested with the same enzymes. The correctness of the cloned DNA sequence was confirmed by sequencing (Institute of Biochemistry and Biophysics, Warsaw, Poland).
For silencing of SGT1 (A and B isoform) the sense: 5 -CACCGCACAGTATTATTGTCAAA GAGTGTGCTGTCCTCTTTGACAATAATACTGTGCTTTTT-3 and antisense: 5 -GCATAAAA AGCACAGTATTATTGTCAAAGAGGACAGCACACTCTTTGACAATAATACTGTGC-3 oligonucleotides comprising the target sequence 5 -GCACAGTATTATTGTCAAAGA-3 , corresponding to a fragment of SGT1, were synthetized, hybridized and the double strand product was cloned into the modified pPUR plasmid (Takara Bio Inc., Shiga, Japan), containing the U6 promoter for RNA Pol III, using BspMI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) [19] to obtain the pPURhU6-SGT1 plasmid. Correctness of the cloning was confirmed by sequencing as described above. To obtain HEK293 cell line stably overexpressing SGT1 (A and B isoform), cells at 75% confluency were transfected with p3×FLAG-CMV-10-SGT1A and p3×FLAG-CMV-10-SGT1B plasmids or p3×FLAG-CMV-10 plasmid (control). After 24 h, cells were treated with geneticin (Thermo Fisher Scientific; Waltham, MA, USA) at a final concentration of 500 ng/mL. The morphology of cells was monitored every day under the light microscope (TMS, Nikon, Melville, NY, USA) and the medium, supplemented with geneticin, was changed every 2 days. Finally, cells were maintained in medium containing 250 ng/mL geneticin. HEK293 cells with overexpression of SGT1 (both isoforms) and control ones were transiently transfected with pcDNA4-α-synuclein-3×FLAG or pcDNA4-3×FLAG plasmid (control) as described above and analyzed 24 h later.
In order to silence SGT1 (A and B isoform), HEK293 cells were transiently transfected with pPURhU6-SGT1 plasmid (encoding shRNA against mRNA for SGT1) or pPURhU6-Luc4 plasmid (encoding shRNA against Renilla Luciferase mRNA) as a control, using Lipofec-tamine2000 as described above. Then, 24 h later, these cells were transfected with pcDNA4α-synuclein-3×FLAG or pcDNA4-3×FLAG and examined after an additional 24 h.

Preparation of Protein Lysates
To obtain cytoplasmic and nuclear fractions, cells were rinsed with ice-cold PBS, (Sigma-Aldrich, St. Louis, MO, USA) collected and lysed according to the Nuclear and Cytoplasmic extraction kit procedure, NE-PER (Thermo Fisher Scientific, Waltham, MA, USA). To obtain total protein lysate cells were lysed in RIPA buffer (Millipore, Burlington, MA, USA), passed 20 times through syringe and incubated on ice for 30 min. Protein lysates were centrifuged at 12,000× g for 20 min at 4 • C. The supernatant was collected and protein concentration was measured by Bradford's method (Bio-Rad, Hercules, CA, USA). A total of 30 µg of protein lysate was taken, precipitated in 1 mL ice-cold acetone and kept at −20 • C until use.

Western Blot Analysis
Proteins precipitated in ice-cold acetone were centrifuged at 20,000× g for 15 min at 4 • C, mixed with Laemmli's sample buffer, incubated at 95 • C for 5 min and subjected to SDS-PAGE according to Laemmli [21]. SDS-PAGE and transfer onto nitrocellulose membrane were carried out as described by Góral et al. [22]. In order to avoid non-specific binding of antibodies, the nitrocellulose was incubated in 5% w/v lyophilized fat-free milk in TBS-T for 1 h at room temperature. Then, the membrane was incubated overnight at 4 • C with primary mouse anti-α-synuclein (Abcam; Cambridge, UK, cat no ab1903) or rabbit anti-SGT1 (BD Biosciences, San Jose, CA, USA) antibodies, both diluted 1:1000. Afterwards, the nitrocellulose was washed in TBS-T and allowed to react for 1 h in room temperature with secondary anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or anti-rabbit IgG (Millipore, Burlington, MA, USA) antibodies conjugated with horseradish peroxidase (HRP) diluted 1:10,000. The level of β-actin, detected by mouse monoclonal antibody conjugated with HRP (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10,000, was used as a reference of protein loading for total cell lysate and for cytoplasmic fraction. The level of PARP, detected by rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000, was used as a reference for the nuclear fraction. The intensities of protein bands on an X-ray film (Kodak, Rochester, NY, USA) were quantified using a manual gel and film documentation in an Ingenius densitometer (Syngene, Frederick, MD, USA) and the Gene Tools software (Syngene, Frederick, MD, USA). Statistical analysis was performed as described below (Section 2.9).

Fluorescence-Based Quantification of Nuclear-Cytoplasmic Localization
Quantifications were performed according to [23]. The intensity of fluorescent staining, which reflects α-synuclein localization, was calculated in selected region of interest (ROI) using the ImageJ 1.42q software (NIH, Bethesda, MD, USA) for both nucleus and cytoplasm. Then, the mean fluorescence value for nucleus and cytoplasm was calculated for 20 cells taken from three independent experiments.

Real Time/Quantitative PCR (RT-qPCR)
Total RNA was isolated from HEK293 cells using the Extractme kit (Blirt DNA, Gdansk, Poland). RNA (2 µg) was then reverse-transcribed using M-MLV Reverse Transcriptase and random nonamers (Sigma-Aldrich, St.Louis, MO, USA). Relative mRNA levels were measured by RT-qPCR using the SYBRGreen system (Thermo Fisher Scientific, Waltham, MA, USA) with 18S rRNA as a standard. The primers used are listed in Table 2. The results were analyzed by absolute quantification with a relative standard curve and normalized to 18S rRNA using the comparative ∆∆Ct method. left for 18 h and MTS assay (Promega, Madison, WI, USA) was performed according to manufacturer's protocol. The level of formazan was measured by recording changes in the absorbance at 490 nm using a microplate reader (Tecan, Morrisville, NC, USA). Viability of cells overexpressing 3×FLAG-SGT1 (SGT1A and SGT1B) or 3×FLAG alone was compared to the viability of those treated with solvent alone. Table 2. List of primers used in RT-qPCR experiments.

Gene
Forward Reverse

Statistical Analysis
Experiments were performed at least in triplicates and results are presented as means ± SEM. Results obtained from analysis of fluorescence intensity, Western blots and RT-qPCR were analyzed by Student's t-test using Microsoft Excel. Results of the MTS assay were analyzed by one-way ANOVA. The level of statistical significance was set at * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

SGT1 Protects HEK293 Cells from Rotenone-Induced Toxicity
At first, we checked whether SGT1, as a co-chaperone of Hsp90, has an effect on the viability of HEK293 cells treated with rotenone, an agent that evokes behavioral and histopathological symptoms similar to those observed in PD [24]. For that, the MTS assay was performed. As it can be seen in Figure 1, SGT1 overexpression (left panel) increases cell viability while silencing (right panel) has no effect. Moreover, overexpression of SGT1 protects HEK293 cells against rotenone-induced toxicity (Figure 1, left panel). On the other hand, rotenone-treated cells with silenced SGT1 were less viable than rotenone-treated control cells (Figure 1, right panel). The mean rates of SGT1 overexpression or silencing (shown in Figure 2A,B, upper panels) calculated from three different cell transfections were 71% ± 11.3% and 49% ± 5.5%, respectively.

SGT1 Affects α-Synuclein Subcellular Localization in HEK293 Cells
To get more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in control HEK293 cells and in cells with altered level of SGT1 (Figure 2A,B, upper panels). For that we applied immunocytochemistry with the use of anti-SGT1 and anti-α-synuclein antibodies. It was found that in control cells α-synuclein was predominantly localized in the nucleus while in cells overexpressing SGT1, α-synuclein was mainly present in the cytoplasm ( Figure  2A, middle panels) in which it could be detected in SGT1-containing complexes (Figure 2A, middle panels) in which it could be detected in SGT1-containing complexes ( Figure  S1). In order to confirm this finding, we analyzed HEK293 cells with diminished level of SGT1. We found that in these cells α-synuclein was predominantly present in the nucleus ( Figure 2B, middle panels). Statistical analysis of fluorescence intensities in the cytoplasmic and nuclear areas was performed in parallel and the results are shown in Figure  2A,B, lower panels.

SGT1 Affects α-Synuclein Subcellular Localization in HEK293 Cells
To get more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in control HEK293 cells and in cells with altered level of SGT1 (Figure 2A,B, upper panels). For that we applied immunocytochemistry with the use of anti-SGT1 and anti-α-synuclein antibodies. It was found that in control cells α-synuclein was predominantly localized in the nucleus while in cells overexpressing SGT1, α-synuclein was mainly present in the cytoplasm (Figure 2A, middle panels) in which it could be detected in SGT1-containing complexes ( Figure S1). In order to confirm this finding, we analyzed HEK293 cells with diminished level of SGT1. We found that in these cells α-synuclein was predominantly present in the nucleus ( Figure 2B, middle panels). Statistical analysis of fluorescence intensities in the cytoplasmic and nuclear areas was performed in parallel and the results are shown in Figure 2A,B, lower panels.
To confirm the immunocytochemistry results, subcellular fractionation and Western blot analysis were performed. As it can be seen in Figure 3, overexpression of SGT1 leads to an increase in α-synuclein level in the cytoplasmic fraction and to a decrease in the level of this protein in the nuclear fraction when compared with control cells.
To confirm the immunocytochemistry results, subcellular fractionation and Western blot analysis were performed. As it can be seen in Figure 3, overexpression of SGT1 leads to an increase in α-synuclein level in the cytoplasmic fraction and to a decrease in the level of this protein in the nuclear fraction when compared with control cells. Notably, silencing of SGT1 did not cause any statistically significant differences in α-synuclein protein level either in the cytoplasm or in the nucleus (Figure 4).  Notably, silencing of SGT1 did not cause any statistically significant differences in α-synuclein protein level either in the cytoplasm or in the nucleus (Figure 4).
To confirm the immunocytochemistry results, subcellular fractionation and Western blot analysis were performed. As it can be seen in Figure 3, overexpression of SGT1 leads to an increase in α-synuclein level in the cytoplasmic fraction and to a decrease in the level of this protein in the nuclear fraction when compared with control cells. Notably, silencing of SGT1 did not cause any statistically significant differences in α-synuclein protein level either in the cytoplasm or in the nucleus (Figure 4).

Altered SGT1 Level Triggers Changes in PINK1 and PARK9 Gene Expression
Previously, it was shown that nuclear α-synuclein is involved in transcriptional regulation of various genes [6,10]. Since SGT1 alters subcellular localization of α-synuclein we checked, by applying RT-qPCR, whether it influences expression of PD-related genes such as PINK1 and PARK9. As it is shown in Figure 5, expression of these genes is lower in SGT1 overexpressing cells, while in those with silenced SGT1, the expression of PARK9 remains the same and that of PINK1 is higher than in control cells.

Altered SGT1 Level Triggers Changes in PINK1 and PARK9 Gene Expression
Previously, it was shown that nuclear α-synuclein is involved in transcriptional regulation of various genes [6,10]. Since SGT1 alters subcellular localization of αsynuclein we checked, by applying RT-qPCR, whether it influences expression of PDrelated genes such as PINK1 and PARK9. As it is shown in Figure 5, expression of these genes is lower in SGT1 overexpressing cells, while in those with silenced SGT1, the expression of PARK9 remains the same and that of PINK1 is higher than in control cells.

Discussion
In this work we have found that SGT1 protected HEK293 cells from rotenoneinduced toxicity and observed that altered level of SGT1 had an effect on α-synuclein subcellular localization in HEK293 cells. More precisely, we found that in control cells the protein was predominantly localized in the nucleus while in cells with overexpressed SGT1 the majority of α-synuclein was present in the cytoplasm. Interestingly, SGT1 homolog, CacyBP/SIP, was also found to protect cells from rotenone-induced toxicity [17] but altered level of CacyBP/SIP had no effect on α-synuclein subcellular localization in HEK293 cells (not shown). α-Synuclein is predominantly a cytoplasmic protein, although some studies pointed to its nuclear localization [4][5][6]. Moreover, earlier results showed that in neuroblastoma SH-SY5Y cells and Drosophila brain cells nuclear αsynuclein was more toxic than cytoplasmic α-synuclein [4]. Thus, it is possible that the beneficial effect of SGT1 on rotenone-treated HEK293 cells may, at least in part, be due to its ability to influence cellular localization of α-synuclein. A positive result of the PLA experiment, which serves to detect molecules located in close proximity, suggests that SGT1 and α-synuclein likely co-exist in the same protein complexes ( Figure S1). Therefore, it is conceivable that SGT1 may engage α-synuclein in cytoplasmic complexes and prevent its accumulation in the nucleus.
The results we have obtained show also that there is a correlation between the nuclear level of α-synuclein and expression of PD-related genes such as PINK1 and PARK9. This is in agreement with other studies showing that nuclear α-synuclein caused alterations in gene expression. For instance, microarray analysis pointed to 15 genes that were up-and 30 that were down-regulated in neuroblastoma cells overexpressing α-synuclein

Discussion
In this work we have found that SGT1 protected HEK293 cells from rotenone-induced toxicity and observed that altered level of SGT1 had an effect on α-synuclein subcellular localization in HEK293 cells. More precisely, we found that in control cells the protein was predominantly localized in the nucleus while in cells with overexpressed SGT1 the majority of α-synuclein was present in the cytoplasm. Interestingly, SGT1 homolog, Ca-cyBP/SIP, was also found to protect cells from rotenone-induced toxicity [17] but altered level of CacyBP/SIP had no effect on α-synuclein subcellular localization in HEK293 cells (not shown). α-Synuclein is predominantly a cytoplasmic protein, although some studies pointed to its nuclear localization [4][5][6]. Moreover, earlier results showed that in neuroblastoma SH-SY5Y cells and Drosophila brain cells nuclear α-synuclein was more toxic than cytoplasmic α-synuclein [4]. Thus, it is possible that the beneficial effect of SGT1 on rotenone-treated HEK293 cells may, at least in part, be due to its ability to influence cellular localization of α-synuclein. A positive result of the PLA experiment, which serves to detect molecules located in close proximity, suggests that SGT1 and α-synuclein likely co-exist in the same protein complexes ( Figure S1). Therefore, it is conceivable that SGT1 may engage α-synuclein in cytoplasmic complexes and prevent its accumulation in the nucleus.
The results we have obtained show also that there is a correlation between the nuclear level of α-synuclein and expression of PD-related genes such as PINK1 and PARK9. This is in agreement with other studies showing that nuclear α-synuclein caused alterations in gene expression. For instance, microarray analysis pointed to 15 genes that were up-and 30 that were down-regulated in neuroblastoma cells overexpressing α-synuclein [25]. Recent RNA-seq analysis revealed that in pluripotent stem cells with increased α-synuclein level there were 131 up-and 115 down-regulated genes when compared to control cells [26]. Additionally, some other studies have demonstrated that α-synuclein could regulate the expression of transcription factors such as CREB and NFAT [3]. In this work we have found that sequestering of α-synuclein to cytoplasm, caused by overexpression of SGT1, changed the expression of PD-related genes, PINK1 and PARK9. Interestingly, in our previous study we have found that SGT1 level was higher in PD brain [15]. As it is known, PINK1 regulates, directly or indirectly, many signaling pathways involved in PD pathogenesis including mitochondrial and Ca 2+ homeostasis, protection against damage of misfolded proteins, autophagy/mitophagy and endoplasmic reticulum-mitochondria crosstalk [27]. In turn, PARK9 encodes a lysosomal ATPase, the mutations and deficit of which have been linked to an early-onset form of PD. Interestingly, it was found that elevated expression of this ATPase reduced α-synuclein toxicity [28].
Altogether, based on the results presented in this work we suggest that SGT1-dependent changes in α-synuclein subcellular localization and PINK1 and PARK9 gene expression strongly argue for the involvement of SGT1 in the pathogenesis of PD, however, more studies are required to elucidate its role in signaling pathways leading to PD development.

Conclusions
In this work we show that SGT1 protects cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. Moreover, we found that in control HEK293 cells α-synuclein is predominantly localized in the nucleus while in cells with elevated SGT1 level it is mainly present in the cytoplasm. Additionally, we discovered that altered SGT1 level, most probably through regulating α-synuclein partitioning between cytoplasm and nucleus, influences expression of PD related genes, PINK1 and PARK9. Overall, our results point to SGT1 as an important factor that may be involved in the pathogenesis of PD.