Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway

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NEUROSCIENCE 1

RESEARCH ARTICLE J. H. Won et al. / Neuroscience xxx (2018) xxx–xxx

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Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway

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Jong Hoon Won, Hyung Jun Jeon, Seok Kyun Kim, In Chul Shin, Ji Min Jang, Hae Chan Ha, Moon Jung Back and Dae Kyong Kim *

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Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-Ku, Seoul 06974, South Korea

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Abstract—Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release. Ó 2019 Published by Elsevier Ltd on behalf of IBRO.

Key words: synaptosomal-associated protein 25, ceramide, neutral sphingomyelinase 2, dopamine, neurotransmission.

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INTRODUCTION

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Neurotransmitters are released from vesicles into the extracellular milieu via exocytosis. This process involves the fusion of intracellular fluid-filled vesicles with the plasma membrane, resulting in the incorporation of vesicle proteins and lipids into the membrane (Salaun et al., 2004). The presynaptic protein synaptosomalassociated protein 25 (SNAP-25) is a critical component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complex, which is responsible for triggering the synaptic release of neuro-

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transmitters (Washbourne et al., 2002; Stoffel et al., 2005; Rizo and Sudhof, 2012). SNAP-25 is considered to regulate the membrane fusion by forming a tight complex that merges the synaptic vesicle membrane with the plasma membrane (Lovat et al., 2004). SNAP-25 is a target membrane-anchored SNARE attached to the plasma membrane via lipid anchors (Hess et al., 1992; Sollner et al., 1993), and contains four palmitoylated cysteine residues and two coiled coil sequences (Weimbs et al., 1997; 1998). The coiled-coil sequences are essential for the interaction between the vesicle-anchored and target membrane-anchored SNAREs. Ceramide, a bioactive sphingolipid, is required in many biological processes such as proliferation, apoptosis, differentiation, inflammation and reportedly triggers the fusion of lipid bilayers during spontaneous fusion (Kunishima et al., 2006; Bartke and Hannun, 2009; Abdul-Hammed et al., 2010). Ceramide is generated by sphingomyelin hydrolysis, catalyzed by three sphingomyelinases (SMases): acid, neutral (nSMase), and alkaline. The three mammalian nSMases – nSMase1, 2, and 3 – are involved in various types of cellular signaling (Clarke et al., 2006). Particularly, the nSMase2, a membrane-associated enzyme, has been widely studied (Farooqui et al., 2010). Previous studies have shown that nSMase2 is located in the Golgi compartment and plasma

*Correspondence to: D. K. Kim, Department of Environmental and Health Chemistry, Chung-Ang University, College of Pharmacy, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea. E-mail address: [email protected] (D. K. Kim). Abbreviations: BSA, bovine serum albumin; co-IP, coimmunoprecipitation; DAPI, 40 ,6-diamidino-2-phenylindole; DEAE, diethylaminoethanol; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; IP, immunoprecipitation; LC/MS/MS, liquid chromatography and tandem mass spectrometry; nSMase, neutral sphingomyelinase; PBS, phosphate-buffered saline; PLA, proximity ligation assay; PVDF, polyvinylidene difluoride; RPMI, Roswell Park Memorial Institute; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; siRNA, small interfering RNA; SMase, sphingomyelinase; SNAP-25, synaptosomal-associated protein 25; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor. https://doi.org/10.1016/j.neuroscience.2019.08.015 0306-4522/Ó 2019 Published by Elsevier Ltd on behalf of IBRO. 1

Please cite this article in press as: Won JH et al. Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway. Neuroscience (2019), https://doi. org/10.1016/j.neuroscience.2019.08.015

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membrane (Milhas et al., 2010), and trafficking of nSMase2 to the plasma membrane occurs in response to cell confluency (Marchesini et al., 2004), H2O2 (Levy et al., 2006), and tumor necrosis factor (Clarke et al., 2007). The anterograde trafficking of nSMase2 from Golgi to the plasma membrane is required for its signaling functions; however, the regulatory mechanism is poorly understood. In this study, we examined the interaction between SNAP-25 and nSMase2 and examined the role of SNAP-25 as a potential regulator of the SMase pathway in synaptic neurotransmission.

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EXPERIMENTAL PROCEDURES

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Materials

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Dr. Maurine E. Linder (Dept. of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA) kindly provided the cDNA encoding SNAP-25b. The cDNA encoding V5-tagged nSMase2 was generously provided by Dr. Yusuf Hannun (Biochemistry and Molecular Biology, Medical University, Charleston, SC, USA). Bovine brain tissue was purchased from a local slaughterhouse in Seoul, Korea and immediately stored at –80 °C. [3H] Palmitic acid (1.2 Ci/mmol), [3H] dopamine (4 Ci/mmol), and [Nmethyl-14C] SM (48.5 Ci/mmol) were purchased from PerkinElmer (Waltham, MA, USA). The DE52 anion exchange gel was purchased from Whatman (Maidstone, UK). Butyl-ToyopearlÒ hydrophobic resin and the diethylaminoethanol (DEAE)-5PW anion exchange column were purchased from Tosho Co. (Tokyo, Japan). Materials for cell culture were obtained from Life Technologies (Carlsbad, CA, USA). Lipofectamine RNAimax, scrambled, and SNAP-25 Stealth small interfering RNA (siRNA) were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies to mouse monoclonal anti-SNAP-25 (Cat. #sc-20038), antisyntaxin1 (Cat. #sc-12736), anti-nSMase2 (Cat. #sc166637), anti-GAPDH (sc-47724) and nonspecific immunoglobulin Gs (IgGs) from mice (Cat. #sc-2028) and rabbits (sc-2027) were supplied by Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies conjugated to Alexa fluorophores, anti-rat Alexa 488, anti-rabbit Alexa 555, and anti-V5 antibody (MA5-1525) were from Thermo Fisher Scientific, while the horseradish peroxidase-conjugated secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). The crosslinkers, including SulfoNHS-LC-biotin and UltraLink avidin beads, were from Pierce (Rockford, IL, USA). N-Lauroyl-D-erythrosphingosine (C12 ceramide [d18:1/8:0]; C12 Cer), Npalmitoyl-D-erythro-sphingosine (C16 ceramide [d18:1/16:0]; C16 Cer), N-stearoyl-D-erythro-sphingosine (C18 ceramide [d18:1/18:0]; C18 Cer), N-arachidoyl-Derythro-sphingosine (C20 ceramide [d18:1/20:0]; C20 Cer), N-lignoceroyl-D-erythro-sphingosine (C24 ceramide [d18:1/24:0]; C24 Cer), N-nervonoyl-D-erythrosphingosine (C24:1 ceramide [d18:1/24:1(15Z)]; C24:1 Cer), D-glucosyl-ß-1,10 N-palmitoyl-D-erythro-sphingosine (C16 glucosyl(ß) ceramide [d18:1/16:0]; C16 glucosyl-

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0 Cer), and D-glucosyl-ß-1,1 N-stearoyl-D-erythrosphingosine (C18 glucosyl(ß) ceramide [d18:1/18:0]; C18 glucosyl-Cer) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). High-performance liquid chromatography (HPLC)-grade chloroform, methanol, and tetrahydrofuran were purchased from J.T. Baker (Phillipsburg, NJ, USA). HPLC-grade water was purchased from Fisher Scientific, Inc. (Waltham, MA, USA). SNAP-25 and nSMase2 recombinant proteins were purchased from Origene (Rockville, MD, USA).

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Transfection of siRNA in PC12 cells

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PC12 cells (Cat# CRL-1721) were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured in RPMI medium supplemented with 5% fetal bovine serum and 10% horse serum at 37 °C in a 5% CO2 incubator. Cells were transfected with scrambled siRNA (Stealth RNAi Negative Control Medium GC Duplex), SNAP-25 siRNA (Cat# RSS302919, RSS302920 and RSS302921, Invitrogen), or nSMase2 siRNA (Cat# RSS331830 and RSS331831, Invitrogen). Subsequently, 50 nM of indicated siRNA was used for knockdown of target protein. After adding the siRNA mixture, the cells were incubated for 48 h. Lipofectamine RNAimax (Invitrogen) was used for transfection according to the manufacturer’s instructions.

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Immunocytochemistry

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PC12 cells were seeded on poly-D-lysine-coated coverslips. Cells were fixed in 4% paraformaldehyde for 10 min. Penetration was performed with 0.3% Triton X100 for 1 h at room temperature. After brief rinsing, the sections were incubated overnight with primary antibodies at 4 °C as follows: 1:400 mouse anti-SNAP25 (Santa Cruz) and 1:400 rabbit anti-nSMase2 (Santa Cruz) in 5% bovine serum albumin (BSA). This was followed by three 10-min washes with phosphatebuffered saline (PBS) and a 1-h incubation with secondary antibodies (goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594; Invitrogen) at room temperature, each at 1:200 and diluted in 5% BSA. The sections were washed with PBS three times and counter-stained with DAPI. Immunofluorescent images were collected using a confocal microscope (LSM 710 Meta Confocal Microscope, Zeiss, Oberkochen, Germany).

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In situ proximity ligation assay (PLA)

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PC12 cells were grown on poly-d-lysine-coated coverslips. The protocol was adopted from the manufacturer’s instructions (Duolink In Situ kit, OLINK BIOSCIENCE, Uppsala, Sweden). Cells were fixed with 4% PFA for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Then, the cells were blocked for 30 min at 37 °C in Duolink blocking solution. Cells were incubated with mouse antinSMase2 and goat anti-SNAP-25 antibodies (Santa Cruz) for overnight at 4 °C. The cells were washed and then incubated with a Duolink PLA PLUS and MINUS

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probes for 1 h at 37 °C. The anti-mouse PLUS probe was used for detecting nSMase2 and the anti-goat MINUS probe for detecting SNAP-25. The cells were incubated with the ligation solution for 45 min at 37 °C, and then incubated with amplification solution. Images were obtained using a confocal microscope (LSM 710 Meta Confocal Microscope, Zeiss, Oberkochen, Germany).

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Partial purification of nSMase from bovine brains

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Membrane-associated nSMase was prepared using a Triton X-100-extraction method as described previously (Jung et al., 2000; Kim et al., 2010a; b). Briefly, the bovine brain (1.5 kg) was homogenized with 7.5 L of homogenizing Buffer V (50 mM Tris–HCl [pH 7.5], 1 mM ethylenediaminetetraacetic acid [EDTA], 3 mM MgCl2, 50 mM KCl, and 10 mM 2-mercaptoethanol) by a Polytron Homogenizer. The homogenate was centrifuged at 10,000g for 10 min and the supernatants were re-centrifuged at 10,000g at 4 °C for 1 h. The pellets were resuspended in 0.75 L Buffer V and 4 M (NH4)2SO4 was added drop-wise until concentration was adjusted to 0.5 M. This mixture was stirred at 4 °C for 2 h and centrifuged at 40,000g at 4 °C for 1 h. To obtain Triton X100-extractable form of membrane-bound nSMase, the pellets were stirred in 2.5 L Buffer V containing with 0.1% Triton X-100, and centrifuged at 40,000g at 4 °C for 1 h. The extracts were applied to a DEAE-cellulose anion exchange column pre-equilibrated with Buffer A (25 mM Tris–HCl [pH 7.5], 1 mM EDTA, and 10 mM 2mercaptoethanol). The bound proteins were eluted using Buffer A containing 0.5 M (NH4)2SO4 and 0.1% Triton X100. The active fractions were applied to a butylToyopearl column, pre-equilibrated with Buffer A at a concentration of 0.5 M (NH4)2SO4. The bound proteins were eluted using distilled water containing 10 mM 2mercaptoethanol. The pool of active fractions was adjusted to 0.1% using Triton X-100 and sonicated at 4 °C. The sonicated fraction was centrifuged at 100,000g at 4 °C for 1 h. The supernatant was applied to a DEAE-5PW HPLC column (21.5 mm  15 cm; Tosho Co., Tokyo, Japan), previously equilibrated with Buffer A containing 0.1% Triton X-100. The bound proteins were eluted by a linear gradient of Buffer A containing 0.25 M (NH4)2SO4 and 0.1% Triton X-100. The active fractions were applied to a Phenyl-5PW HPLC column (21.5 mm  15 cm; Tosoh Co.) previously equilibrated with Buffer A containing 0.5 M (NH4)2SO4. The bound proteins were eluted using a linear gradient of distilled water containing 10 mM mercaptoethanol. To apply to a Sephacryl S-300 gel filtration column, the active fractions were concentrated with a CentriconÒ filter (Vivascience, Epsom, UK). The concentrated fraction was applied to the gel filtration column (30 mm  60 cm) previously equilibrated with Buffer A containing 0.1 M NaCl. Each fraction was assayed for nSMase activity.

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Isolation of synaptosome and extraction of membrane protein The synaptosome fraction was isolated from the rat striatum by centrifugation using a Percoll gradient

3

(Dunkley et al., 2008). Briefly, rat striatum tissue was homogenized in 0.32 M sucrose buffer (0.32 M sucrose, 1 mM EDTA, and 250 lM DTT; pH 7.4) with 10 strokes of a Dounce homogenizer and centrifuged at 1000g for 10 min at 4 °C. The supernatant fraction was collected and directly applied to a discontinuous Percoll gradient, which comprised of four layers of 3% (vol/vol), 10% (vol/ vol), 15% (vol/vol), and 23% (vol/vol) Percoll. The gradient was then centrifuged at 31,000g for 10 min at 4 °C. Synaptosome fractions were collected and diluted with PBS. The diluted synaptosome fractions were recentrifuged to remove Percoll, re-suspended in assay buffer (1.25 mM NaCl, 1.5 mM KH2PO4, 5 mM KCl, 1.25 mM CaCl2, 1.5 mM MgSO4), and centrifuged at 5000g for 10 min at 4 °C to completely remove residual sucrose. The final pellets were sonicated in 100 mM Tris–HCl (pH 7.5) containing 0.1% Triton X-100 (Jung et al., 2000), and gently shaken for overnight at 4 °C. After extraction step, the fraction was centrifuged at 100,000g and the supernatant was used for analysis. For extracting membrane protein, PC12 cells were washed three times with ice-cold PBS and then sonicated in 100 mM Tris–HCl (pH 7.5) containing 0.1% Triton X100 and a protease inhibitor. The homogenate was centrifuged at 2000g for 10 min at 4 °C to pellet the cellular debris. The lysate was centrifuged at 100,000g to obtain the membrane fraction. The pellet was sonicated in 100 mM Tris–HCl (pH 7.5) containing 0.1% Triton X100, gently shaken for 2 h at 4 °C for membrane protein extraction, and then centrifuged at 100,000g. The supernatant was then removed for analysis. All procedures and animal treatments were performed according to the guidelines for laboratory animal experimentation set by Chung-Ang University. This work was approved by the Chung-Ang University Ethical Committee related to the use of laboratory animals.

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Biotinylation of cell surface protein

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The cells were seeded in poly-D-lysine-coated 6-well plates. The surface protein was labeled with Sulfo-NHSLC-biotin as described previously (Loder and Melikian, 2003; Holton et al., 2005). Briefly, the cells were washed with ice-cold PBS (pH 7.4) containing 1.0 mM MgCl2 and 0.1 mM CaCl2 (PBS2+). The washed cells were incubated with Sulfo-NHS-LC for 30 min at 4 °C. The residual SulfoNHS-LC was quenched by 100 mM glycine in PBS2+ and the cells were washed three times with ice-cold PBS2+. After the wash, cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors. The biotinylated proteins were separated using streptavidin beads, which were recovered by centrifugation. Proteins bound to the beads were eluted in sample buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 0.01% BPB, 6% glycerol) and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.

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Immunoprecipitation (IP) and immunoblotting

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Striatal synaptosomes or PC12 cells were lysed with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1% Triton X-100 and protease inhibitors at 4 °C for 1 h. To discard

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A

B 3.0

6

2.5

5

2.0

0.1% 0.5 M

1.5

3

1.0

2

0.5

1

0 0 2 4 6 8 10 12 14 Fraction Number

C

0

2.5 2.0 1.5 1.0 0.5 0 0

D

0.1% 0.5 M

10 20 30 40 50

0

20 40 60 80 Fraction Number

0.5 M

0 5 10 15 20 25 30 35 40

0

Fraction Number

0

0

Fraction Number 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

0

0

4.0 3.5 3.0

E

4

8 7 6 5 4 3 2 1 0

0.5 M

0 10 20 30 40 50 60 Fraction Number

0

100

Fig. 1. Chromatographic profiles of Triton X-100-extractable Mg2+-dependent membrane-associated nSMase from bovine brain. A membrane-associated neutral form of nSMase was extracted with 0.5 M (NH4)2SO4 and 0.1% Triton X-100 from the pellets of bovine brain homogenates centrifuged at 10,000g and purified by sequential column chromatography as described in Section 2.6. The graphs show SMase activity from the (A) DEAE-cellulose anion exchange column (bed volume of DE52 gel, 2.0 L), (B) butyl-Toyopearl column (bed volume of 150 mL), (C) DEAE-5PW HPLC column (21.5 mm  15 cm; Tosoh Co.), (D) Phenyl-5PW HPLC column (21.5 mm  15 cm; Tosoh Co.), and (E) Sephacryl S-300 gel filtration column (30 mm  60 cm). The solid lines in all figures indicate UV absorbance.

debris, the lysed samples were centrifuged for 10 min at 2000g. Each indicated antibody was mixed with the proteins and incubated for overnight at 4 °C. For immunoprecipitation of the recombinant proteins, the samples were mixed with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.02% Triton X-100 for 4 h. Each indicated antibody was mixed with the protein samples and incubated for overnight at 4 °C. Immunoprecipitated proteins were pelleted and washed three times with 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The IP supernatants and IP pellets were used for nSMase activity and immunoblotting. The samples were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA in Tris-buffered saline (50 mM TrisHCl, 150 mM NaCl, 0.2% Tween 20) for 1 h. The blocked membranes were probed with the indicated primary antibody for overnight. After three washes, the membranes were incubated with the horseradish peroxidaseconjugated secondary antibody for 2 h. The protein bands were detected using ECL Western Blotting Substrate (Pierce).

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In vitro nSMase activity assay

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Total protein (30 mg) was used for the nSMase activity in assay solution (100 lL) containing 10 mM MgCl2, 2.5 lM [Nmethyl-14C] SM (approximately 30,000 cpm), 2 mM sodium deoxycholate, and 100 mM Tris– HCl (pH 7.5). Reactions were performed at 37 °C for 10 min and then terminated by adding 320 lL of chloroform/methanol (1:1 volume) and 30 lL of 2 N HCl

Table 1. Purification of nSMase from ‘‘Triton X-100 extracts” obtained from bovine brain tissue Step

Protein (mg)

Total Activity (nmol/min)

Specific activity (nmol/min/mg)

Purification (fold)

Yield (%)

Tx100 Extract DE52 Butyl DEAE Phenyl SS-300

9525 768 138 39.1 4.2 1.47

3285.12 2083.64 1695.21 332.13 112.21 48.29

0.34 2.71 12.28 8.49 26.72 32.85

8 36 25 77 95

63.43 51.6 10.11 3.42 1.47

Please cite this article in press as: Won JH et al. Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway. Neuroscience (2019), https://doi. org/10.1016/j.neuroscience.2019.08.015

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A Fraction No.

19

20

21

22

23

24

25

26

nSMase2

75 kDa

SNAP-25

25 kDa

B

C IP supernatant

Mouse IgG supernatant anti-SNAP-25 Ab supernatant

Time (h)

0

8

24

Mouse IgG pellet anti-SNAP-25 Ab pellet

nSMase2

75 kDa

SNAP-25

25 kDa

nSMase activity (cpm)

2500 2000 1500

*

*

* IP pellet

1000

Time (h) *

*

500

*

0 0

8

16 Time (h)

24

0

8

24

nSMase2

75 kDa

SNAP-25

25 kDa

Fig. 2. Analysis of protein–protein interaction between SNAP-25 and nSMase2 in bovine brain. (A) Representative western blot of the nSMase2 and SNAP-25 in fractionation of the purification of Triton X-100-extractable nSMase from bovine brain. (B) The active nSMase fraction from Sephacryl S-300 was used for IP. Anti-SNAP-25 or mouse IgG was added, and the mixture was incubated for the indicated times. The immunoprecipitates were analyzed for SNAP-25 by immunoblotting. The immunoprecipitation pellets were washed, and aliquots of immunoprecipitates were assayed for nSMase activity. (C) The supernatant and immunoprecipitates were separated by 15% SDS-PAGE, transferred to PVDF membranes, and probed with an anti-nSMase2 or SNAP-25 antibody.

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(Bligh and Dyer, 1959). After vortexing, the upper phase (200 mL) was transferred into 2.5 mL scintillation solution (Insta-Gel-XF, Packard Instrument Co., Meriden, CT, USA). Radioactivity was determined using a Packard Tri-carb liquid-scintillation counter.

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Analysis of ceramide levels in PC12 cells

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The level of ceramide was determined as described previously (Jeon et al., 2005). Briefly, PC12 cells (2  105), cultured in 6-well plates, were labeled with 1 mM [3H] palmitic acid (1 mCi/mL; PerkinElmer) for 24 h. The cells were washed and lysed in 400 mL of acidified methanol solution (CH3OH:0.5-N HCl = 1:1), followed by addition of 200 mL of chloroform. The samples were vortexed for 20 min and then centrifuged at 15,000g for 30 min at 4 °C. The chloroform phase was collected and dried under a nitrogen stream in a new tube. To dissolve the dried lipids, 20 mL of a chloroform–methanol solution (1:1; v/v) was added and thin-layer chromatography was used for separating non-radioactive

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ceramides in a solvent system containing chloroform: methanol:acetic acid:water (21.25:1.125:1.25:0.125, respectively; v/v). Ceramide spots were detected by iodine vapor and radioactivity was measured using a bscintillation counter (Tri-carb 1600 TR; Packard Instrument Co.).

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Quantification of ceramide species by liquid chromatography and tandem mass spectrometry (LC/ MS/MS)

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For calibration, lipid standards were prepared as described previously (Won et al., 2018). Each lipid was dissolved in methanol at a final concentration of 200 lg/ mL and this solution was diluted in methanol with 0.1% formic acid at several concentrations. C12 ceramide was added to each sample as internal standard. For lipid extraction and preparation, the modified Bligh-Dyer method was used (Bligh and Dyer, 1959; Yoo et al., 2006) and the ceramide species were quantified by LC/ MS/MS as previously described (Akundi et al., 2005).

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A

B

Synaptosome

PC12

75kDa

IB: nSMase2

75kDa

IB: SNAP-25

25kDa

IB: SNAP-25

25kDa

10000

3000

Immunoprecipitated nSMase activity (CPMA)

Immunoprecipitated nSMase activity (CPMA)

IB: nSMase2

***

2000

***

1000

***

7500 5000 2500

0

*

0

IP:

IP:

C

D nSMase2 siRNA

Scrambled siRNA

nSMase2

75kDa

GAPDH

37kDa

IB: nSMase2

75kDa

IB: SNAP-25

25kDa

80

40 0

E

**

IB: nSMase2

75kDa

IB: SNAP-25

25kDa

**

6000 **

**

4000

**

2000 0

Immunoprecipitaed nSMase activities (CPMA)

120

Immunoprecipitaed nSMase activities (CPMA)

nSMase activities (% of Control)

** 8000

***

4000 3000

**

2000 1000 0

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Dopamine release assay

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The dopamine release assay was performed as described previously (Bloch-Shilderman et al., 2002). PC12 cells were seeded onto poly-D-lysine-coated 6-well plates at a density of 2.5  105/well, 24 h before performing the assay. The medium was replaced with fresh RPMI1640 to equilibrate for 30 min at 37 °C, followed by addition of 60 nM [3H] dopamine (1 lCi/mL) and incubation for 20 min at 37 °C. After loading of [3H] dopamine, the cells were washed once with a serum-supplemented medium and twice with a serum-free medium containing 1 mM ascorbic acid. The medium was added and preincubated for 30 min and then A23187 (calcium ionophore) was added. Each sample (200 mL) was centrifuged for 10 min at 1000g. The released dopamine was determined by measuring radioactivity in the supernatants. The total [3H] dopamine uptake was measured based on the radioactivity in lysed cells.

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Transient co-transfection of SNAP-25b and V5nSMase2 cDNAs in MCF-7 cells MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 37 °C and 5% CO2 incubator. For surface level analysis, MCF7 cells (5  105) were seeded into 6-well plates. After 24 h, the cells were transfected with 2.5 lg SNAP-25b and 2.5 lg V5-nSMase2 cDNA using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The control group was transfected with 2.5 lg empty pcDNA3.1 vector and 2.5 lg V5-nSMase2 cDNA. Cells were grown for an additional 24 h after transfection and biotinylation of cell surface proteins was performed. For immunochemical analysis, MCF-7 Cells (0.5  105) were seeded into a 35-mm confocal dish. The cells were transfected with 0.625 lg SNAP-25b and 0.625 lg V5-nSMase2 cDNA using Lipofectamine 2000. The control group was transfected with 0.625 lg empty pcDNA3.1 vector and 0.625 lg V5-nSMase2 cDNA. Antibody penetration enhancement and all other procedures were performed as described in Section 2.4.

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All data are expressed as the mean ± standard error (SE). SigmaStat 3.5 Systat Software (San Jose, CA, USA) was used for statistical analysis. Statistical differences between groups were analyzed by one-way analysis of variance with subsequent Tukey’s tests. In all cases, a p-value <0.05 was considered statistically

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significant. Exact numbers for all experiments are provided in the figure legends.

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SNAP-25 tightly binds to nSMase2, including in the partially purified fraction from bovine brain

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The nSMase2 enzyme in the Triton X-100 extract was partially purified by sequential use of anion exchange, hydrophobic interaction chromatography, anion exchange HPLC, hydrophobic HPLC, and gel filtration fast protein liquid chromatography as described previously (Kim et al., 2010a; b). The purification profile is shown in Fig. 1. Table 1 summarizes the chromatography and nSMase purification process. The obtained data suggested that nSMase2 forms complexes with other proteins. The partially purified active fraction was analyzed for protein distribution by SDS-PAGE. Although the chromatographic process resulted in 95-fold purification of nSMase2 from the Triton X-100 extracts of the bovine brain, SNAP-25 was also enriched in the nSMase2containing fractions (Fig. 2A). Further, the anti-SNAP-25 antibody immunoprecipitated SNAP-25 protein in the Sephacryl S-300 active fractions. The nSMase activity in the anti-SNAP-25 antibody pellet increased in a timedependent manner upon incubation with anti-SNAP-25 antibody (Fig. 2B). The IP pellet incubated with mouse IgG (negative control) did not show nSMase activity (Fig. 2B). The supernatant and pellet of the IP-mixture was separated using 15% SDS-PAGE and immunoblotted with anti-SNAP-25 antibody. The anti-SNAP-25 antibody pellet contained nSMase2 (Fig. 2C). Taken together, these data suggest that nSMase2 tightly binds SNAP-25.

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SNAP-25 binds to nSMase2 in the synaptosome and PC12 cells

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In a previous study, we suggested that nSMase is involved in membrane disturbance related to the release of dopamine (Jeon et al., 2005). Additionally, neurotransmitter release is mediated by vesicle SNAREs (vSNAREs), synaptobrevin/vesicle-associated membrane protein, and the target SNARES (t-SNAREs) syntaxin and SNAP-25 in presynaptic nerve terminals (Sudhof and Rizo, 2011). Therefore, we hypothesized that SNAP-25 complex might interact with SMase. We next investigated the protein–protein interaction between SNAP-25 and nSMase2 via co-immunoprecipitation (coIP) experiments using synaptosomal preparations from mouse striatum. We performed co-IP experiments with a Triton X-100-soluble fraction from the synaptosome frac-

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3 Fig. 3. Analysis of protein–protein interaction between SNAP-25 and nSMase2 in mouse synaptosomes and PC12 cells. A Triton X-100-soluble nSMase fraction was used for IP in rat synaptosomes (A) and in PC12 cells (B). nSMase2 and SNAP-25 were immunoprecipitated as described in Section 2.8 and the immunoprecipitates were analyzed for nSMase2 and SNAP-25 by immunoblotting. The immunoprecipitation pellets were washed, and aliquots of the immunoprecipitates were assayed for nSMase activity. (C–D) Scrambled and nSMase2 siRNA were transfected into PC12 cells. After 48 h, the nSMase2 level was investigated (C) and the IP pellets were washed, after which the aliquots of the IP pellet were assayed for nSMase activity (D). IP pellets were analyzed for nSMase2 and SNAP-25 by immunoblotting in mixture of recombinant SNAP25 and nSMase2 (E). Ab: antibody. The results represent the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01.

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tion. The Triton-X 100 soluble fraction was incubated with anti-SNAP-25 antibody or anti-nSMase2 antibody, or alternatively with nonspecific mouse or rabbit antibodies, which were used as the respective negative controls in this experiment. As a result, nSMase2 was detected with the anti-SNAP-25 antibody in the IP pellet from synaptosomes (Fig. 3A) and PC12 cells (Fig. 3B). In the nSMase activity assay, the IP pellet of the anti-SNAP-25 antibody showed a higher level of nSMase activity than the IP pellet of the anti-nSMase2 antibody (Fig. 3A and B). The IP pellet of nSMase2 did not contain SNAP-25 in synaptosomes (Fig. 3A) or PC12 cells (Fig. 3B). To verify that the nSMase activity of the anti-SNAP-25-precipitated pellet was due to nSMase2, we knocked down nSMase2 by siRNA transfection in PC12 cells. Transfection of nSMase2 siRNA significantly decreased the nSMase activity in PC12 cells (Fig. 3C). Furthermore, nSMase activity and protein were lost in the anti-SNAP-25 IP pellet when compared with the scrambled siRNA transfection group (Fig. 3D). In order to further clarify the interaction between SNAP-25 and nSMase2, we examined the binding of recombinant nSMase2 and SNAP-25 proteins. In the IP pellet of the anti-SNAP-25 antibody, nSMase2 was detected and showed nSMase activity (Fig. 3E). Taken together, these results demonstrate that SNAP25 and nSMase2 interact in the synaptosome and PC12 cells. SNAP-25 is required for nSMase2 plasma membrane localization We investigated the role of SNAP-25 in nSMase2 function. First, we examined whether SNAP-25 directly affects nSMase activity. PC12 cells were transfected with scrambled or SNAP-25 siRNA and then nSMase activity was analyzed. Transfection of SNAP-25 siRNA significantly decreased SNAP-25 expression, but did not affect nSMase activity (Fig. 4A). Next, we investigated the role of SNAP-25 in nSMase2 trafficking. Transfection of SNAP-25 siRNA significantly reduced the cell surface level of nSMase2 (Fig. 4B). Our previous study showed that nSMase2 induced Ca2+dependent generation of ceramide (Kim et al., 2010a; b). To determine whether SNAP-25-dependent nSMase2 translocation is involved in Ca2+-dependent ceramide generation, PC12 cells were treated with A23187. In SNAP-25 siRNA-transfected cells, A23187-induced nSMase2 translocation was significantly inhibited (Fig. 4C). We examined the location of SNAP-25 and nSMase2 in PC12 cells. As shown in Fig. 5A, SNAP-25 was colocalized with nSMase2 in the plasma membrane. We also observed colocalization of SNAP-25 and nSMase2 signals in the plasma membrane in an in situ PLA (Fig. 5B). In a previous study, MCF-7 cells were used to

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investigate the trafficking of nSMase2 (Milhas et al., 2010). SNAP-25a is the predominant isoform during the embryonic stage, while SNAP-25b becomes abundant after postnatal development. Especially, SNAP-25b is the predominant isoform in the brain (Bark et al., 1995; Daraio et al., 2018). Thus, we performed co-transfection of V5-tagged nSMase2 and SNAP-25b in MCF-7 cells to examine whether nSMase2 trafficking is regulated by SNAP-25 expression. Localization of nSMase2 was investigated by immunofluorescence and cell surface biotinylation as described under Materials and Methods. SNAP-25b overexpression increased the cell surface level of V5-nSMase2 in MCF-7 cells (Fig. 5C and D). Moreover, V5-nSMase2 colocalized with giantin, a Golgi marker, only in V5-nSMase2-overexpressing MCF-7 cells (Fig. 5D). Taken together, our results suggest that SNAP25b is involved in the anterograde trafficking of nSMase2 from the Golgi to the plasma membrane.

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SNAP-25 is required for ceramide production

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We examined the relationship between SNAP-25 and nSMase2 in ceramide production. The ceramide level was examined in SNAP-25 siRNA- and A23187-treated PC12 cells with an isotope, as described in Section 2.10. In scrambled siRNA-transfected cells, the levels of ceramide increased in a dose-dependent manner by A23187 treatment, reaching levels that were approximately two-fold higher after treatment with 5 lM A23187 for 30 min. In SNAP-25 siRNA-transfected cells, ceramide formation was reduced compared with that in scrambled siRNA-transfected cells (Fig. 6A). We next used LC/MS/MS to compare the ceramide formation in SNAP-25 or nSMase2 siRNA transfected cells. To exclude the possibility that ceramide was produced in a de novo pathway, we applied FB1, an inhibitor of de novo synthesis (Wang et al., 1991). Transfection of nSMase2 siRNA significantly decreased nSMase2 expression (Fig. 6B). As shown in Fig. 6C, A23187induced ceramide production was reduced in SNAP-25 siRNA-transfected cells, compared with that in scrambled siRNA-transfected cells. The reduced ceramide level in SNAP-25 siRNA-transfected PC12 cells was similar to that in nSMase2 siRNA-transfected cells (Fig. 6C). Notably, the sphingomyelin level was increased in SNAP-25 siRNA-transfected cells compared with that in scrambled siRNA-transfected cells (Fig. 6D). These results indicate that SNAP-25 is involved in nSMase2-mediated ceramide production.

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Dopamine release was regulated via ceramide generation pathway in PC12 cells

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SNAP-25 is involved in the docking of synaptic vesicles to the target membrane (Mohrmann et al., 2013). As shown

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3 Fig. 4. Effect of SNAP-25 siRNA on nSMase2 translocation in PC12 cells. The cells were transfected with scrambled siRNA or SNAP-25 siRNA. After transfection, the cells were washed and were lysed by sonication with buffer. Cell lysates were used for immunoblotting and nSMase assay (A). After transfection, the cell surface was biotinylated to determine the nSMase2 surface levels (B). After A23187 treatment for the indicated times, nSMase2 translocation was evaluated by western blotting of biotinylated proteins with an anti-nSMase2 antibody (C). The results represent the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01.

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in Fig. 7A, A23187-induced dopamine release was reduced in SNAP-25 siRNA-transfected cells compared with the scrambled siRNA-transfected cells. Next, we investigated whether the sphingomyelin pathway (SM pathway) affects dopamine release. Previously, treatment of cell-permeable ceramide was shown to stimulate dopamine release (Jeon et al., 2005). In PC12 cells, transfection of nSMase2 siRNA did not alter dopamine release (data not shown). However, co-treatment with FB1 and nSMase2 siRNA transfection decreased dopamine release compared with the scrambled siRNA-transfected cells (Fig. 7B). These results suggest that ceramide produced through the de novo pathway and the SM pathway is involved in exocytosis. To determine whether ceramide regulates dopamine release, cell-permeable C6-ceramide was added to SNAP-25 siRNA-transfected PC12 cells. As shown in Fig. 6C, dopamine release was reduced to approximately 75% in SNAP-25 siRNA-transfected cells in presence of A23187. Treatment with C6-ceramide compensated for the reduced dopamine release by up to 90% (Fig. 7C and D) in SNAP-25 or nSMase2 siRNAtransfected cells. In the absence of A23187, single treatments with C6-ceramide did not significantly modify the dopamine release (Fig. 7C and D), which is consistent with the results of a previous study (Jeon et al., 2005) The nSMase2 activity is regulated in a Ca2+-dependent manner (Kim et al., 2010a; b) Moreover, the Ca2+ionophore A23187 induced ceramide production in a concentration- and time-dependent manner, which paralleled the dopamine release. This increase was significantly reduced when 1 mM of EGTA was added (Jeon et al., 2005). Collectively, these results suggest that SNAP-25 is involved in ceramide production, which mediates exocytosis down-stream of the associated calcium influx and nSMase activation.

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DISCUSSION

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The nSMase activity is reported to be highest in the brain, suggesting that nSMase plays a role in brain-specific processes (Chatterjee et al., 1999). However, this highly specific activity of nSMase does not clarify its role in neuronal processes such as apoptosis, differentiation, survival, and ischemic cell death. In this study, we investigated the functional link between SNAP-25 and nSMase2. The dopaminergic neurons form widely arborized axons in striatum (Matsuda et al., 2009). The nSMase2 protein is enriched in the brain (Hofmann et al., 2000). We used previously described column chro-

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matographic methods to purify the membrane-associated, magnesium-dependent nSMase (Jung et al., 2000). Our results suggested that SNAP-25 binds to nSMase2, even when it was partially purified from the bovine brain (Fig. 2). Thus, we hypothesized that SNAP-25 exists as a stable complex with not only the SNARE proteins, but also nSMase2. Furthermore, immunoprecipitation and nSMase activity assays, with and without knockdown of nSMase2 (Fig. 3), indicated that SNAP-25 and nSMase2 interact with each other. SNAP-25 is a component of SNARE protein complex, which is involved in membrane fusion (Kumar et al., 2015; Zhou et al., 2015). The SNARE assembly acts centrally to drive membrane fusion for neurotransmitter release, including dopamine. In the cytoplasm, vesicleassociated membrane protein-2 engages with two tSNAREs, syntaxin 1A and SNAP-25, to form a core complex bridging two membranes (Jahn and Scheller, 2006). Sphingolipid is a major component of the plasma membrane, which is the main site of nSMase2dependent ceramide formation. nSMase2 recycling is important for regulating its catalytic activity (Clarke et al., 2008). Based on these, we hypothesized that SNAP-25 might be involved in the translocation of nSMase2. Our colocalization studies showed that SNAP-25 and nSMase2 were present together at the plasma membrane (Fig. 5A and B), and SNAP-25 knockdown reduced the nSMase2 surface levels (Fig. 4B and C). Furthermore, overexpression of SNAP-25 increased nSMase2 translocation to the plasma membrane (Fig. 5C and D). Interestingly, Ca2+-dependent ceramide production was reduced in SNAP-25 siRNA-transfected cells (Fig. 6A and B). In Fig. 6C, knockdown of nSMase 2 reduced the levels of the short-chain, but not the long-chain ceramides. On the other hand, knockdown of SNAP-25 caused reduction of both long- and short-chain ceramides. These observations may imply that an additional isoform of SMase is linked to the SNAP-25 complex. nSMase2 siRNA transfection (data not shown) or use of FB1, an inhibitor of de novo ceramide synthesis, did not alter dopamine release (Jeon et al., 2005). However, nSMase2 siRNA transfection in the presence of FB1 decreased dopamine release in PC12 cells (Fig. 7B). It has been reported that external treatment with ceramide increased exocytosis in PC12 cells (Tang et al., 2007). In our study, addition of C6-ceramide compensated for the reduced dopamine release in SNAP-25 or nSMase2

3 Fig. 5. nSMase2 translocation regulated by SNAP-25 expression. PC12 cells were immunostained with polyclonal goat anti-SNAP-25 (green: labeled with anti-rat Alexa Fluor 488) and polyclonal rabbit anti-nSMase2 (red: labeled with anti-rabbit Alexa Fluor 555) antibodies. Arrows represent the colocalization of nSMase2 and SNAP-25 (A). An in situ PLA using proximity probes against nSMase2 and SNAP-25 was performed to visualize SNAP-25/nSMase2 heterodimers in PC12 cells. PC12 cells were stained with anti-nSMase2 mouse monoclonal antibody (1:200) and anti-SNAP-25 goat polyclonal antibody (1:200). Signals were detected with a Duolink detection kit 613 (red), and nuclei were counterstained with DAPI (B). MCF-7 cells (0.5  105) were seeded in 35-mm confocal dishes and 24 h later were transiently transfected with indicated cDNA (0.625 mg/dish) as described in Section 2.13. The surface was biotinylated to determine the nSMase2 surface levels and probed for the indicated protein (C). Control is cells transfected with V5-tagged nSMase2 and SNAP-25b indicates the cells of co-transfected with V5-tagged nSMase2 and SNAP-25b. The transfected cells were fixed and stained with anti-V5 (green) and anti-giantin (red) as described in Section 2.3. (D) The results are representative of three independent experiments that each produced similar results. The results represent the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Scale bars: 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Won JH et al. Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway. Neuroscience (2019), https://doi. org/10.1016/j.neuroscience.2019.08.015

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Fig. 6. Analysis of ceramide species level in PC12 cells. PC12 cells were plated into 6-well culture dishes and the transfection complex was diluted with serum-free medium and added directly to the cells; this sample was added to normal medium after 3 h. (A) Cells were incubated for 12 h and labeled with 1 mM [3H] palmitic acid for 24 h. A23187 was added after washing with free-RPMI1640 medium. After 30 min, cells were collected and the levels of ceramide were determined. PC12 cells were transfected with scrambled, SNAP-25 or nSMase2 siRNA and were treated with the FB1 (10 lM, 1 h) and A23187 (0.5 lM, 30 min) 48 h later. (B) The nSMase2 expression level was analyzed in scrambled or nSMase2 siRNA. (C) The levels of ceramide subspecies were measured by LC/MS/MS. (D) Indicated siRNA were transfected into PC12 cells; 48 h later, the levels of sphingomyelin species were investigated. The results represent the mean ± SE of three independent experiments. *p < 0.05, #p < 0.01.

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siRNA-transfected cells (Fig. 7C-D). Therefore, SNAP-25 may be involved in the production of ceramide that mediates exocytosis. Exocytosis involves membrane fusion between the synaptic vesicles and plasma membrane. Ceramide is known to trigger fusion of lipid bilayers during spontaneous fusion (van Meer and Holthuis, 2000; Hoekstra et al., 2003). C6-ceramide has two aliphatic chains like long-chain physiological ceramide (Stover and Kester, 2003), and cone-shaped ceramide induces a curved membrane (Venkataraman and Futerman, 2000; Utermohlen et al., 2008). This plasma membrane perturbation provides a means of cellular uptake and endocytosis. In addition, ceramide works as a substrate

for enzymes like ceramide kinase and ceramidase, which regulate membrane fusion. Ceramide-1-phosphate generated by ceramide kinase regulates membrane fusionrelated events via liposome fusion (Won et al., 2018) and sphingosine by ceramidase, which regulates vesicle fusion and trafficking (Dressler and Kolesnick, 1990; Hinkovska-Galcheva et al., 1998; Mitsutake et al., 2004; Rohrbough et al., 2004). Synaptic transmission is regulated by catecholamine neurotransmitter; particularly, dysregulation in dopamine neurotransmission causes various neurological and psychiatric diseases such as drug addiction, schizophrenia, and Parkinson’s disease (Self and

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Fig. 7. Inhibition of ceramide formation pathway suppressed DA release in PC12 cells. (A, B) The cells were transfected with scrambled, SNAP-25, or nSMase2 siRNA. After transfection, fresh RPMI 1640 was added, and the cells were equilibrated for 30 min at 37 °C. The cells were then loaded with [3H] DA and incubated. The medium in the cultures was changed to fresh media and pre-incubated for 30 min. A23187 was added and DA release was measured as described in Section 2.12. (C, D) After transfection of SNAP-25 siRNA or nSMase2 siRNA, the cells were treated with C6ceramide (5 mM) for 15 min in the presence or the absence of 1 mM A23187 (C, D) and DA release was measured. The results are representative of three independent experiments showing similar results. *p < 0.05.

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Nestler, 1995). Some studies have shown that SNAP-25 is involved in mental diseases and secretory diseases such as schizophrenia, Parkinson’s disease, and diabetes (Nagamatsu et al., 1999; Zhang et al., 2002; Thompson et al., 2003; Eguiagaray et al., 2004). Additionally, sphingolipids are involved in mental diseases (Schmitt et al., 2004). Understanding sphingolipid metabolism and SNAP-25 function may provide insights that will aid in the development of medicines for diabetes, schizophrenia, Parkinson’s disease, and depression. Here, we demonstrated that SNAP-25 is involved in ceramide production and exocytosis. Based on our results, SNAP-25 is required for nSMase2 translocation and ceramide production at the plasma membrane. nSMase was reported to be mainly localized to the membrane (Milhas et al., 2010). Although SNAP-25 does not affect the total nSMase2 levels or activity, SNAP-25 siRNA results

revealed lower ceramide levels in the cells. This suggests that the nSMase2 localization is important for ceramide production via the SM pathway. Impairments in exocytosis are related to severe secretory diseases and elucidating exocytosis mechanisms is essential for developing improved medications for these diseases.

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ACKNOWLEDGEMENTS

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This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (NRF-2015M3A9C7030121 and NRF2017M3A9D8048414).

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CONFLICT OF INTEREST

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The authors have no conflicts of interest to declare. Please cite this article in press as: Won JH et al. Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway. Neuroscience (2019), https://doi. org/10.1016/j.neuroscience.2019.08.015

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AUTHOR CONTRIBUTIONS

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Jong Hoon Won, Hyung Jun Jeon and Seok Kyun Kim conceived and designed the experiments. Jong Hoon Won, In Chul Shin, Ji Min Jang, Hae Chan Ha, and Moon Jung Back performed the experiments. Jong Hoon Won and Dae Kyong Kim analyzed the data. Jong Hoon Won wrote the manuscript.

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(Received 19 March 2019, Accepted 7 August 2019) (Available online xxxx)

Please cite this article in press as: Won JH et al. Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway. Neuroscience (2019), https://doi. org/10.1016/j.neuroscience.2019.08.015

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