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By an unknown mechanism, alpha-synuclein (α-syn) inhibits autophagy in yeast and human cells. Herein, using the yeast Saccharomyces cerevisiae, we tested the hypothesis that α-syn disrupts autophagy by inhibiting the required association of sorting nexin 4 (Snx4) with phagophores. Snx4 contains a phox (PX) homology domain that selectively binds membranes enriched in phosphatidylinositol 3-phosphate (PI3P). Using fluorescence microscopy, we show that upon nitrogen starvation, 70% of the cells exhibited green puncta (phagophores); whereas identically treated cells expressing α-syn exhibited a significantly lower percentage of cells (30%) with such puncta. Our interpretation is that α-syn outcompetes Snx4 for binding to membranes enriched in PI3P, resulting in fewer phagophores and consequently inefficient induction of autophagy. As a control, we tested whether α-syn disrupts the binding of Vps27-GFP to late endosomes/multivesicular bodies (MVBs). Vps27 contains a PI3P-binding domain called FYVE. α-Syn did not disrupt the binding of Vps27-GFP to late endosomes. α-Syn likely inhibits the binding of PX- but not FYVE-containing proteins to PI3P because FYVE domains bind more than two-orders of magnitude tighter than PX domains. We propose that in all cells, whether yeast or human, α-syn has the potential to inhibit protein trafficking pathways that are dependent on PX-domain proteins such as sorting nexins.

We tested the ability of alpha-synuclein (α-syn) to inhibit Snx3-retromer-mediated retrograde trafficking of Kex2 and Ste13 between late endosomes and the trans-Golgi network (TGN) using a Saccharomyces cerevisiae model of Parkinson’s disease. Kex2 and Ste13 are a conserved, membrane-bound proprotein convertase and dipeptidyl aminopeptidase, respectively, that process pro-α-factor and pro-killer toxin. Each of these proteins contains a cytosolic tail that binds to sorting nexin Snx3. Using a combination of techniques, including fluorescence microscopy, western blotting and a yeast mating assay, we found that α-syn disrupts Snx3-retromer trafficking of Kex2-GFP and GFP-Ste13 from the late endosome to the TGN, resulting in these two proteins transiting to the vacuole by default. Using three α-syn variants (A53T, A30P, and α-synΔC, which lacks residues 101–140), we further found that A53T and α-synΔC, but not A30P, reduce Snx3-retromer trafficking of Kex2-GFP, which is likely to be due to weaker binding of A30P to membranes. Degradation of Kex2 and Ste13 in the vacuole should result in the secretion of unprocessed, inactive forms of α-factor, which will reduce mating efficiency between MATa and MATα cells. We found that wild-type α-syn but not A30P significantly inhibited the secretion of α-factor. Collectively, our results support a model in which the membrane-binding ability of α-syn is necessary to disrupt Snx3-retromer retrograde recycling of these two conserved endopeptidases.

The protein alpha-synuclein (α-syn) is unusual because, depending on its conformation and the type of cell in which it is expressed, it is pro-death or pro-survival, triggering neurodegeneration in Parkinson’s disease and enhancing cell survival of some melanomas. To probe the function of α-syn in melanoma, we used CRISPR/Cas9 to knockout SNCA, the gene that codes for α-syn, in SK-Mel-28 melanoma cells. The SNCA-knockout clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species and proliferated slower than control cells. These SNCA-knockout clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. In the excised SNCA-knockout xenografts, TfR1 decreased 3.3-fold, ferritin increased 6.2-fold, the divalent metal ion transporter 1 (DMT1) increased threefold, and the iron exporter ferroportin (FPN1) decreased twofold relative to control xenografts. The excised SNCA-KO tumors exhibited significantly more ferric iron and TUNEL staining relative to the control melanoma xenografts. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn.

Malignant melanoma is one of the most aggressive and fifth most prevalent cancer among men and women. Clinical and epidemiological studies shows that Parkinson’s disease (PD) patients have higher risk of developing invasive melanoma, and, reciprocally, patients with invasive melanoma have a higher risk of developing PD. Alpha-synuclein (α-syn; SNCA), an intrinsically disordered protein expressed in neurons, is also highly expressed in malignant melanoma and is a common molecular signature between Parkinson’s disease and melanoma. We have previously reported that SK-Mel-28 SNCA-knockout (KO) cells exhibit suppressed tumor growth and proliferation in vivo and in vitro, and that putting back SNCA via lenti virus restored the phenotype to that of the control cells. The present study is designed to investigate the role of α-syn in melanoma progression in SK-MEL-28 cells in the context of metastasis and the epithelial-to-mesenchymal transition (EMT). The SK-Mel-28 SNCA-KO clones showed a significant decrease in the cell-surface protein L1CAM, a major cell adhesion molecule highly expressed in various melanoma and correlates with melanoma progression, whereas the knock in clones resembled the control. Migration and invasion assay were performed using transwell chambers showed a profound migratory and invasive defect in SNCA-KO clones. To explore further whether this is a proliferative or migratory defect, a single cell phagokinetic motility assay was performed with colloidal gold which showed a marked decrease in cell motility in knockout clones compared to control and knock in clones, which was consistent with our migration and invasion experiments. To determine the proteins involved in the inhibition of invasion mediated by α-syn, we immunoblotted for the mesenchymal markers N-cadherin and vimentin, and these were significantly downregulated in the SNCA-KO clones. These data reinforce that knocking out SNCA impedes melanoma cell migration and invasion in SK-Mel- 28 cell line, implying that α-syn might be a novel therapeutic target for malignant melanoma. Citation Format: Nithya Gajendran, Santhanasabapathy Rajasekaran, Nirjhar M. Aloy, Sahar Shekoohi, Stephan N. Witt. Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2443.

Background Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. Results We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. Conclusions In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes. Electronic supplementary material The online version of this article (10.1186/s12896-018-0479-1) contains supplementary material, which is available to authorized users.

Figure S2. Immunostained cross sections of testis at 3-, 4- and 8- week-old adult testis. α-Smooth muscle actin (α-Sma) is a myoid cell marker. Collagen 1 and Laminin 5 mark the ECM and β1 integrin marks the periphery of seminiferous tubules. Scale bar 50 μm. For 3 right panels of the 4 week time point, scale bar 100 μm. (PDF 6157 kb)

Figure S1. Distribution of transduction efficiency of AAVs in mouse testis. A) Immunostaining with EdU staining. Edu incorporation and staining in 4 to 5 weeks old males reveals proliferating sperm progenitors in the seminiferous tubules outlined by Laminin 5 staining. B) Top: Wholemount of dissected testis imaged for live GFP. Bottom: Cryosection immunostained for GFP. Scale bars 100 μm. C) Histogram representing the transduction efficiency of the various serotypes. For each testis, the number of GFP+ cells in a 10X field was enumerated; the number GFP+ cells / 0.58 mm2 (mean ± standard deviation; AAV5, 0; AAV8, 26 ± 14.1; AAV9, 104 ± 24.1; AAV9 S499A, 45.3 ± 10.4; AAVrh10, 78 ± 15.1; n = 3 animals; n = 2 animals for AAV 8). (PDF 2291 kb)

The Parkinson’s disease-associated protein alpha-synuclein (α-syn) is often highly expressed in melanoma. α-Syn appears to exert a pro-survival function in melanoma cells, possibly by maintaining autophagy homeostasis. The long-term goal of our work is to determine the mechanism by which α-syn confers survival in melanoma cells. Herein, we used CRISPR (clustered regularly interspaced short palindromic repeats) to disrupt SNCA, the gene that codes for α-syn, in the melanoma cell line SK-Mel-28. Western blotting and real-time-qPCR verified that we generated four α-syn knockout (KO) clones. Three clones showed no expression of α-syn, whereas one had about 50% less α-syn than the control cells. DNA sequencing is consistent with three of the clones having both alleles of SNCA knocked out, whereas one clone has only one allele knocked out. α-Syn KO clones were tested in a variety of assays to ascertain the function of α-syn. First, the percentage of cells with exofacial phosphatidylserine (PS) was determined by annexin V-FITC/PI staining in conjunction with fluorescence-activated cell sorting. On average, there was a statistically significant 50% increase in exofacial PS (annexin V+/PI-) in three of the four α-syn KO clones compared to control cells. Exofacial PS was increased in the α-syn KO clones without a concomitant increase in apoptotic cells (annexin V+/PI+). Second, the clones were tested for growth in vivo using SCID mice. All four clones showed a ~65% reduction in the rate of tumor growth, followed over 12 weeks. Third, immunohistochemical staining of excised tumors from the SCID mice showed that the proliferation marker Ki-67 was significantly (p< 0.0001) lower in the tumors generated from the KO clones compared to tumors from control cells. In conclusion, given that α-syn avidly binds to PS, one possibility is that α-syn is like molecular Velcro that binds to PS molecules embedded in the inner leaflet, and this binding helps ATP-driven flippases keep PS in the inner leaflet. Loss α-syn then increases exofacial PS, which is a marker of senescence. Our working model is that α-syn is an anti-senescence protein, and that the α-syn KO clones grow slower than control cells in vivo because the clones have entered a senescent state. Citation Format: Sahar Shekoohi, Santhanasabapathy Rajasekaran, Shu Yang, Sureshbabu Nagarajan, Dhaval Patel, Xiuping Yu, Stephan N. Witt. Knocking out the gene (SNCA) that codes for the Parkinson’s disease-related protein alpha-synuclein in SK-Mel-28 melanoma cells significantly retards tumor growth in SCID mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 896.