A delicate redox state balance is maintained in cells by the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and the antioxidant system that detoxifies them. In a balanced redox state, a low concentration of ROS/RNS is maintained, and ROS/RNS can activate the specific signaling pathways that are required for diverse cellular functions, including cell growth and immune responses. However, overproduction of ROS/RNS or decreased antioxidant capacity can disrupt the redox balance, causing oxidative/nitrosative stress. ROS and RNS are highly reactive free radicals, and one such ROS/RNS is nitric oxide (NO). NO plays important roles in the regulation of neuronal, immune, and cardiovascular systems and is produced in many mammalian cells through a reaction catalyzed from L-arginine by a family of NO synthases. NO has an unpaired electron in its pi molecular orbital, which can react with, for example, proteins, lipids, and DNA. S-nitrosylation, a post-translational modification of cysteine residues on specific proteins, regulates protein function and is the result of the reaction between NO and the protein residues. Protein S-nitrosylation has been found in the pathogenesis of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, and is known to contribute to the formation of the intracellular inclusion bodies associated with neurodegenerative diseases. For example, S-nitrosylation of Parkinson was found to affect its enzymatic activity and to lead to the Parkinsonian phenotype. Lafora disease (LD, OMIM 254780) is a fatal autosomal recessive neurodegenerative disorder. LD initially manifests during the teenage years with generalized tonic-clonic seizures, myoclonus, absences, drop attacks, and visual hallucinations, and patients usually die within 10 years of the first symptoms. LD is caused by mutations in Epilepsy, Progressive Myoclonus Type 2A or 2B (EPM2A or EPM2B), which encode Laforin, a glucan phosphatase, or Malin, an E3 ubiquitin ligase, respectively. LD is characterized by the accumulation of insoluble glucans, like glycogen, called Lafora bodies (LBs) in the cytoplasm of cells from most tissues. Mutations in EPM2A and EPM2B account for about 80% of the LD families that were screened for genetic lesions. In the remaining families, the role for a third gene was suggested. Because protein S-nitrosylation has been found in the pathogenesis of neurodegenerative diseases, S-nitrosylation of Laforin may be a third factor in LD pathogenesis. We hypothesized that S-nitrosylation of Laforin may affect its enzymatic activity and lead to LD; similar to the finding that S-nitrosylation of Parkin affects its enzymatic activity and leads to Parkinson’s disease. To test this hypothesis, we first determined whether Laforin is S-nitrosylated, using a biotin switch assay, and whether the function of Laforin is affected by S-nitrosylation. Furthermore, we observed LB formation in cells expressing wild-type Laforin S-nitrosylated by an NO donor.

Lafora disease (LD, OMIM 254780) is a neurodegenerative disease characterized by the accumulation of insoluble glucans called Lafora bodies (LBs) and is caused by mutations in genes that encode the glucan phosphatase, Laforin, or the E3 ubiquitin ligase, Malin. We aimed to test whether S-nitrosylation of Laforin affects its phosphatase activity and is thus involved in the disease.

Laforin is S-nitrosylated by an NO donor

We first tested whether Laforin was S-nitrosylated by an NO donor using a biotin switch assay. Lysates from HEK293 cells transfected with wild-type Laforin were treated with or without CysNO. After the treatment, free thiol groups were blocked and SNO groups were biotinylated. S-nitrosylated proteins were isolated on avidin beads. Both unmodified and S-nitrosylated Laforin were detected by western blotting. As shown in figure 1A, Laforin was 2.4 times more S-nitrosylated in response to the NO donor, which indicates that Laforin was S-nitrosylated by NO. Error bars in the graph represent the standard deviation (SD) of 6 independent experiments.

Glucan-binding capacity of S-nitrosylated Laforin is maintained

We examined whether Laforin functions were affected by its S-nitrosylation under the same conditions used to test for S-nitrosylation by NO (see Fig. 1A). The buildup of LBs is caused by loss of Laforin function. In normal glycogen metabolism, an enzymatic error of glycogen synthases can cause phosphate to be incorporated into glycogen. When phospho-glycogens are produced in this way, Laforin and other glycogen phosphorylases bind to glycogen and remove the phosphate. After the dephosphorylation reaction, Laforin dissociates from glycogen by binding to Malin and can then be degraded by a proteasomal system. Loss of function of Laforin and/or Malin can result in the accumulation of unremoved phosphates or Laforin, which disrupts the intricate spherical structure of glycogen and leads to a buildup of LBs. Therefore, glucan- and Malin-binding activity and the phosphatase activity of Laforin are all required to avoid LB accumulation. To examine whether any of these three functions of Laforin are affected by S-nitrosylation, we compared the functions of unmodified and S-nitrosylated Laforin. For glucan-binding, lysates from HEK293 cells transfected with wild-type Laforin or glucan-binding dead Laforin mutant (W32G) were treated with or without CysNO and incubated with glucan-conjugated beads. Glucan-bound Laforin was analyzed by western blotting. As shown in figure 1B, similar amounts of both S-nitrosylated Laforin and unmodified Laforin were bound to the glucan. Error bars in the graph represent the SD of 5 independent experiments. These results suggest that the glucan-binding capacity of S-nitrosylated Laforin was similar to that of unmodified Laforin.

Malin-binding capacity of S-nitrosylated Laforin is maintained

For Malin-binding, lysates from HEK293 cells transfected with wild-type Laforin and wild-type Malin were treated with or without CysNO and co-immunoprecipitated. Malin-binding of Laforin was analyzed by western blotting. As shown in figure 1C (i), similar amounts of both S-nitrosylated Laforin and unmodified Laforin bound to Malin. These results suggest that the Malin-binding capacity of S-nitrosylated Laforin is similar to that of unmodified Laforin. Importantly, N-ethylmaleimide-sensitive factor (NSF) did not co-immunopreciptate Malin, indicating that the assays were performed correctly (Fig. 1C(ii)).

S-nitrosylation of Laforin inhibits its phosphatase activity

For phosphatase activity, GST-tagged wild-type Laforin and a phosphatase dead Laforin mutant (C266S; Cys is the catalytic residue) expressed in E. coli were purified and treated with or without CysNO. After treatment, phosphatase activity was measured using pNPP as a substrate. As shown in figure 1D (i), the phosphatase activity of Laforin decreased by 80% in response to the NO donor. Error bars in the graph represent the standard error of the means (SEM) of 3 independent experiments in triplicates. To test whether the decreased phosphatase activity was caused by S-nitrosylation, we used a chemical-reducing agent. Previous reports suggested that protein S-nitrosylation was reversible, and the declined enzymatic activity caused by the NO donor could be reversed to the basal level by a chemical-reducing agent. The declined phosphatase activity of wild-type Laforin was reversed to 60% of the basal level after incubation with the chemical-reducing agent 2-mercaptoethanol (2-ME), indicating that S-nitrosylation of Laforin was reversible (Fig. 1D(ii)). These results suggest that S-nitrosylation of Laforin inhibits its phosphatase activity.

LB formation in cells expressing wild-type Laforin is increased by NO

We observed LB formation in cells expressing wild-type Laforin S-nitrosylated by the NO donor. LBs are seen in most cell types. In this study, we used the HeLa cell line to observe LB formation. The HeLa cells expressing wild-type Laforin were prepared on coverslips and incubated in the presence or absence of GSNO. The number of cells expressing wild-type Laforin and the number of those cells with LB were counted. A representative image of untreated or treated wild-type Laforin-expressing cells is shown in figure 1E (i). LBs are insoluble glucans and Laforin can bind to LBs because of its glucan-binding capacity. Inclusion bodies in figure 1E (indicated by white arrows) are Laforin-binding aggregates and may be LBs. As shown in figure 1E (ii), the cells treated with NO contained 1.4 times more LBs than the untreated cells. Error bars in the graph represent the SD of 4 independent experiments.

We hypothesized that Lafora disease (LD) may be caused by S-nitrosylation of Laforin because we know that Parkinson’s disease can be caused by S-nitrosylation of Parkin. To test this hypothesis, we first determined whether Laforin was S-nitrosylated using the biotin switch assay and whether the three main functions of Laforin were affected by S-nitrosylation. Furthermore, we observed LB formation in cells expressing wild-type Laforin S-nitrosylated by NO. For the first time, in this study, we showed that Laforin was S-nitrosylated by the NO donor and S-nitrosylation of Laforin inhibited its phosphatase activity. Furthermore, we observed that LB formation in cells expressing wild-type Laforin was increased by the NO donor and endogenous NO. LB formation is caused by loss of Laforin phosphatase activity. Based on our results, we propose a possible hypothesis to describe LD pathogenesis; namely, the decrease in Laforin phosphatase activity by S-nitrosylation leads to increased LB formation. Laforin has nine cysteine residues that can be S-nitrosylated. We speculated that the S-nitrosylation site may be C266 (the catalytic residue) because S-nitrosylation of Laforin remarkably inhibited its phosphatase activity. To determine the S-nitrosylation sites, Laforin mutants with individual cysteine residues replaced with serine were constructed. These mutants were tested to determine whether the mutant Laforin could be S-nitrosylated (Suppl. Fig. F). However, contrary to our expectation, the C266S mutant Laforin was more S-nitrosylated than the wild-type Laforin. This result suggests that replacement of C266 with serine generated artificial S-nitrosylation sites, which did not allow us to determine the S-nitrosylation site. Further work is needed to determine the Laforin S-nitrosylation sites. LD is a fatal autosomal recessive neurodegenerative disorder with generalized tonic-clonic seizures and myoclonus. LD is caused by mutations in EPM2A or EPM2B, which encode Laforin and Malin, respectively. Mutations in EPM2A and EPM2B account for about 80% of the LD families that have been screened for genetic lesions. In the remaining families, the role for a third gene has been suggested. Our results suggest that LD is caused not only by mutations in EPM2A and EPM2B, but also by the S-nitrosylation of Laforin. We propose that S-nitrosylation of Laforin could be a third factor for LD.

Our data show that the phosphatase activity of Laforin is reduced by nitric oxide, suggesting that LD phenotypes can be caused by nitrosative stress.

Cell culture experiments should be repeated in animal models or neurons.

Test if endogenous NO would affect LB formation.

Test if NO treatment would affect degradation of Laforin-Malin complex.

Test if NO treatment would affect ubiquitination of Laforin.

Antibodies and reagents

The antibodies and reagents we used in this study were: Hoechst 33342, trihydrochloride, trihydrate, Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen, Carlsbad, CA, used 1:100), anti-Myc tag antibody (clone 9E10, used 1:1000 for western blotting, 1:100 for immunofluorescence), stabilized goat anti-mouse IgG (H+L), peroxidase conjugated (Pierce, Rockford, IL, used 1:1000), and anti-HA tag antibody (clone 16B12, Covance, Princeton, NJ, used 1:1000).

Cell culture and transfection

Human embryonic kidney (HEK293) cells and human cervix epitheloid carcinoma (HeLa) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 08458-74, Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% FBS (Cat. 171012, Lot. 9B0148, Nichirei Biosciences Inc., Tokyo, Japan), in 5% CO2 at 37°C. Transient transfection was performed using transfection reagent PEI Max (1 µg/mL, Polysciences, Inc., Warrington, PA). DNA, PEI Max, and serum-free DMEM were mixed in a ratio of 1 µg: 2 µL: 25 µL. The mixture was incubated at room temperature for 15 min and then added to the cells. The cells were cultured in 5% CO2 at 37°C for 1 or 2 overnights.

Plasmids

To construct plasmids for the expression of Laforin, pcDNA-LDH+His/Myc was provided by Dr. Yamakawa (RIKEN Brain Science Institute, Saitama, Japan). Myc-tagged Laforin was subcloned into pQCXIP (Clontech Laboratories, Inc, Mountain View, CA) or pGEX6P1 (GE Healthcare, Piscataway, NJ) for expression in mammalian or bacterial cells respectively. Point mutations of Laforin were introduced using a QuikChange kit (Agilent, Santa Clara, CA) or a KOD-Plus-Mutagenesis kit (Toyobo Co., Ltd., Osaka, Japan). Plasmids for the expression of Malin (pcDNA3-HA-Malin) and NSF (pcDNA3-Myc-NSF) were provided by Profs. Carlos Roma-Mateo (Instituto de Biomedicina de Valencia, CSIC and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Valencia, Spain) and Yasuko Iwakiri (Yale University, CT), respectively. All the plasmids used in this study were verified by DNA sequencing.

Biotin switch assay

HEK293 cells were transfected with pQCXIP-Laforin-myc or Laforin mutants. Cells were lysed in lysis buffer (50 mM NaCl, 250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1% (v/v) Triton X-100, 0.1 mM neocuproine). After sonication, the lysates were clarified by centrifugation at 4°C, 11,700 × g for 10 min. The supernatants were collected and their protein concentrations were determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Cat. 500-0006, Bio-Rad, Hercules, CA) and diluted to 0.4 mg of protein solution into 1 mL of HEN buffer (250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1 mM neocuproine). Then 200 µM CysNO (100 µM NaNO2, 100 µM cysteine, 0.5 mM HCl) was added and allowed to react at room temperature for 20 min. After incubation, 4 mL of blocking buffer (HEN buffer + 20 mM methyl methane thiosulfonate (MMTS, Lot. BCBH0692V, Sigma-Aldrich, St. Louis, MO), 2.5% (v/v) SDS) was added at 50°C for 20 min with gentle vortex every 4 min to block free thiol groups. After removing excess MMTS by acetone precipitation, the pellets were re-suspended in 100 µL HENS buffer (HEN buffer + 1% (v/v) SDS) and supplemented with 2 µL of ascorbate solution (50 mM (+)-sodium L-ascorbate (Sigma-Aldrich) in HEN buffer) and 33 µL of biotinylating reagent (4 mM EZ-Link HPDP-biotin (Prod. 21341, Lot. OG188581, Thermo Fisher Scientific, Inc.) in N,N-dimethylformamide) to reduce nitrosothiols to thiols and biotinylate. After removing excess biotin by acetone precipitation, the pellets were resuspended in 225 µL HENS buffer and 10 µL samples were saved for western blot. The remaining suspensions were mixed with 450 µL neutralization buffer (100 mM NaCl, 20 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.5% (v/v) Triton X-100) and the biotinylated proteins were isolated with 20 µL NeutrAvidin UltraLink Resin (Prod. 53150, Lot. LI149138, Thermo Fisher Scientific, Inc.) at 4°C overnight with constant rotation. After washing 3 times with wash buffer (neutralization buffer + 600 mM NaCl), the beads were resuspended in 20 µL neutralization buffer. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.

Glucan-binding assay

HEK293 cells were transfected with pQCXIP-Laforin-myc or Laforin mutants. Cells were lysed in lysis buffer (50 mM Hepes-KOH (pH 7.5), 150 mM KCl, 0.1% (v/v) Triton X-100, 1 mM PMSF, 0.1% beta-mercaptoethanol). After incubation for 5 min on ice, the lysate was clarified by centrifugation at 4°C, 11,700 × g for 10 min. The supernatants were divided into half, and CysNO, at a final concentration of 300 µM, was added to one of the samples for S-nitrosylation in a dark at room temperature for 20 min. After incubation, the samples were incubated with amylose resin (Cat. E8021S, Lot. 319-42; New England Biolabs Inc., Ipswitch, MA) at 4°C for 1 h. Amylose is a glucan that partially shares its structure with glycogen. After washing 3 times with lysis buffer, the sample volume was adjusted to 20 µL. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.

Malin-binding assay

HEK293 cells transfected with pQCXIP-Laforin-myc and pCDNA3-HA-Malin were treated with 100 µM CysNO in 5% CO2 at 37°C for 2 h. Cells were lysed in lysis buffer (50 mM NaCl, 250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1%(v/v) Triton X-100, 0.1 mM neocuproine, 1 mM PMSF). After incubation for 5 min on ice, the lysates were clarified by centrifugation at 4°C, 11,700 × g for 10 min. For assay, the supernatants were incubated with Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (Sigma-Aldrich; Cat. A7470, Lot. 093M4823) at 4°C for 2 h. After washing 3 times with lysis buffer, the sample volume was adjusted to 20 µL. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.

Western blotting

The western blot samples were fractionated by SDS-PAGE using a suitable composition of polyacrylamide gel and transferred to a PVDF membrane (Merck Millipore Ltd., Darmstadt, Germany). The membrane was blocked with 4% skim milk in phosphate-buffered saline (PBS) with 1% Tween 20 (PBST) for 1 h and then incubated for 1 h with primary antibodies diluted in 4% skim milk in PBST. After incubation, the membrane was washed 3 or 4 times with PBST for 5–10 min each wash and incubated for 30 min with secondary antibodies conjugated to horseradish peroxidase (HRP). After the membrane was again washed 3 or 4 times with PBST for 5–10 min each wash, it was reacted with AmershamTM ECLTM Western Blotting Detection Reagents (GE Healthcare) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) and target proteins were detected using LAS-4000 (GE Healthcare). Quantification of the western blots was performed using Image J software (https://imagej.nih.gov/ij/).

Protein purification and phosphatase assay

E. coli BL21 (DE3) transformants harboring different glutathione S-transferase (GST)-tagged Laforins were grown in 3–5 mL LB/ampicillin at 37°C overnight. The bacterial culture was transferred to new 500 mL LB/ampicillin and grown at 37°C until the absorbance at 660 nm reached about 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 0.1 mm, and the cultures were maintained overnight at 25°C. Cells were harvested and resuspended in 20 mL sonication buffer (50 mM HEPES-NaOH (pH 7.0), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 2 mM DTT, 2 mM PMSF). Cells were disrupted by sonication and clarified by centrifugation at 4°C, 2,500 × g for 15 min. The supernatants were incubated with 20–30 µL of Glutathione SepharoseTM 4B (Code No. 17-0756-01, Lot. 301195; GE Healthcare). After washing 3 times with sonication buffer, the glutathione beads were resuspended in 100–200 µL of sonication buffer. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Cat. 500-0006, Bio-Rad), SDS-PAGE, and Coomassie Brilliant Blue (CBB) staining using CBB Stain One (Lot. L1K9496, Nacalai Tesque, Inc.). Samples were stored at 4°C. In vitro phosphatase assay was performed using 1.5 µg of the recombinant proteins on glutathione beads. The beads with the proteins were washed once with phosphatase buffer (0.2 M Tris-HCl (pH 6.8), 1 mM DTT) and suspended in 100 µL of phosphatase buffer. Then 100 mM CysNO was added to a final concentration of 200 µM and reacted in the dark at room temperature for 20 min. After washing once with phosphatase buffer and resuspending in 100 µL of phosphatase buffer, 1 M para-nitrophenylphosphate (pNPP, Lot. MOT4381, Nacalai Tesque, Inc.) was added to a final concentration of 150 mM for the phosphatase reaction at 37°C for 5 min. 90 µL of the reaction supernatants were taken and transferred to 96 well plates. The reactions were then stopped by adding 10 µL of 5 M NaOH and the absorbance was measured at 405 nm.

Lafora body assay

HeLa cells on coverslips were transfected with pQCXIP-Laforin-myc. After 14 h of transfection, the coverslips were transferred to fresh culture medium in the presence or absence of 100 µM CysNO or GSNO (50 µM NaNO2, 50 µM reduced glutathione, 0.5 mM HCl) and incubated in 5% CO2 at 37°C for 2 h. LB formation in the cells was observed by immunofluorescence microscopy, as described below. Approximately 40–60 cells expressing wild-type Laforin, and the numbers of those cells with LB, were counted in each experiment.

Immunofluorescence microscopy and image data analysis

HeLa cells on coverslips were fixed with 10% Formalin in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were blocked with 4% bovine serum albumin (BSA) in PBS for 15 min, and then incubated for 15 min with primary antibodies diluted in 4% BSA in PBS. The cells were washed 3 times with PBS, and incubated for 15 min with secondary antibodies conjugated to Alexa fluorophores (Invitrogen). After washing, the coverslips were mounted on microscope slides (Matsunami Glass Ind., Ltd., Osaka, Japan) and the cells were imaged using a microscope (AXIO OBSERVER A1 system, LD A-Plan 32x/0.40 Ph1, Carl Zeiss, Oberkochen, Germany) equipped CCD camera (1392×1040 pixels, Nippon Roper, Tokyo, Japan) or FV1000 confocal microscope system (Olympus, Tokyo, Japan). Image data were processed and quantified using Image J software.

JSPS KAKENHI Grant Number 23570167 and 26440055 of MEXT (Ministry of Education, Sport, Culture, Science and Technology in Japan), and the Futaba Electronics Memorial Foundation to A.S.

We thank Profs. Takashi Uehara (Okayama University), Kazuhiro Yamakawa (RIKEN), and Yasuko Iwakiri and Teruo Utsumi for their kind support with reagents and plasmids, and Toshiko Sunagawa (Okayama University) for her technical assistance.

Not applicable.