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Amyotrophic Lateral Sclerosis Mutant VAPB Causes a Nuclear Envelope Defect 1

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Duvinh Tran, Antonious Chalhoub, Allana Schooley, Wendy Zhang and Johnny K. Ngsee 3

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Neuroscience, Ottawa Hospital Research Institute, Cellular and Molecular Medicine, 5

University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada 6

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Corresponding Author: Johnny K. Ngsee 8

TEL (613) 562-5800 x 2251 9

E-MAIL jngsee@uottawa.ca 10

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Key words: Amyotrophic Lateral Sclerosis, VAPB, Nuclear envelope, gp210, Nup214, 14

Emerin, ERGIC 15

Running title: VAPB Nuclear Envelope Defect 16

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Abbreviations List 18

ALS Amyotrophic Lateral Sclerosis 19

EMD Emerin 20

ER endoplasmic reticulum 21

ERGIC ER-Golgi intermediate compartment 22

FFAT two phenylalanines on an acidic track 23

INM inner nuclear membrane 24

NE nuclear envelope 25

NPC nuclear pore complex 26

Nup nucleoporin 27

ONM outer nuclear membrane 28

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© 2012. Published by The Company of Biologists Ltd.Jo

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Summary 31

A proline to serine substitution (P56S) in VAPB causes an autosomal dominant form of 32

Amyotrophic Lateral Sclerosis (ALS). We show that the mutation also causes a nuclear envelope 33

(NE) defect. Transport of Nucleoporins (Nups) and Emerin (EMD) to the NE is blocked, 34

resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression 35

of the FFAT motif (two phenylalanines on an acidic track) antagonizes this mutant VAPB effect 36

and restores transport to the NE. VAPB function is required for transport to the NE with 37

knockdown of endogenous VAPB recapitulating this phenotype. Moreover, we identified this 38

compartment as ER-Golgi intermediate compartment (ERGIC) with NE membrane proteins 39

transiting to ERGIC before VAPB-dependent retrograde transport to the NE. 40

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Introduction 42

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused 43

by death of motor neurons. Familial ALS8 is caused by a mutation in the VAPB gene 44

(Nishimura et al., 2004; Chen et al., 2010). Overexpresion of the P56S mutation leads to 45

formation of large ER-derived membranes (Nishimura et al., 2004; Kanekura et al., 2006; 46

Teuling et al., 2007; Prosser et al., 2008; Suzuki et al., 2009). VAPB is an integral membrane 47

protein with an N-terminal Major Sperm Protein (MSP) domain and a C-terminal transmembrane 48

domain. The cytoplasmic MSP domain interacts with the FFAT motif (two phenylalanine in an 49

acidic track) (Loewen et al., 2003) found in oxysterol-binding protein (OSBP or ORP) family 50

(Wyles et al., 2002), NIR phospholipid transfer proteins (Lev, 2004), and ceramide transport 51

protein (Kawano et al., 2006). Coordinated membrane recruitment of these proteins by VAPs is 52

thought to regulate lipid composition at membrane contact sites that in turn affects organelle 53

morphology (Amarilio et al., 2005; Peretti et al., 2008). Substitution of Pro56 with Ser exposes a 54

hydrophobic patch (Furuita et al., 2010; Kim et al., 2010) that renders the protein highly prone to 55

aggregation (Kanekura et al., 2006). We have previously shown that co-overexpression of a 56

FFAT-containing fragment but not when the two Phe were substituted with Ala (named AAAT) 57

resolved the abnormal ER morphology induced by mutant VAPB (Prosser et al., 2008). Here we 58

report that the mutation also causes a NE defect characterized by separation of the outer (ONM) 59

and inner nuclear membrane (INM). This defect is caused by disruption of transport of NE 60

proteins as loss of VAPB led to their accumulation in dilated cytoplasmic foci containing 61

ERGIC-53. This suggests that NE proteins transit through ERGIC with VAPB function required 62

for final transit to the NE. 63

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Results and Discussions 65

VAPB-P56S Causes Swelling of the NE 66

To gain insights to the consequences of the mutant protein, we performed an electron 67

microscopic analysis on CHO overexpressing VAPB-P56S or simultaneously overexpressing 68

mutant VAPB and FFAT- or AAAT-containing fragment. Convoluted ER tubules have been 69

observed in HeLa overexpressing mutant VAPB (Teuling et al., 2007; Fasana et al., 2009), and 70

these were also evident in some cells. However, the most prominent features in cells 71

overexpressing mutant VAPB alone or together with AAAT were large cytoplasmic vacuole-like 72

structures and a dilated or “herniated” NE (Fig. 1A-C). The vacuole-like structures likely 73

correspond to dilated mutant VAPB membranes viewed under light microscopy (Kanekura et al., 74

2006; Teuling et al., 2007; Prosser et al., 2008). Interestingly, ~75% of these cells also showed a 75

prominent NE defect characterized by separation of the INM and ONM at discrete regions (Fig. 76

1A, arrows). The gap between the two membranes can be as much as 500 nm apart with a mean 77

of 160 ± 32 nm, which is over two-fold greater than the 70 ± 5 nm gap in control cells (Fig. 1D). 78

Connecting ER tubules when detected also appeared dilated (Fig. 1C, #). These ER and NE 79

defects were not observed in cells co-overexpressing mutant VAPB and the FFAT fragment, 80

consistent with the ability of this motif to neutralize the adverse effects of the mutant protein. 81

Thus, overexpression of mutant VAPB not only causes formation of aberrant cytoplasmic 82

membranes but also results in separation of the two nuclear membranes. 83

Retention of Nup and Emerin (EMD) in Mutant VAPB-Induced Dilated Membranes 84

We next examined mislocalization of Nups as a possible cause since nuclear pores 85

spanning the two nuclear membranes help maintain their close apposition. We examined two 86

Nups with vastly different residence times at the NE and membrane topology: gp210 is a highly 87

dynamic integral membrane protein of the nuclear pore complex (NPC), while Nup214 which 88

forms the structural scaffold has a NE residence time an order of magnitude higher (Daigle et al., 89

2001; Rabut et al., 2004). In empty vector and VAPB-WT transfected cells, gp210-GFP formed a 90

ring-like pattern encompassing the rim of the NE as well as localized to scattered cytoplasmic 91

puncta (Fig. 2A), consistent with previous studies. Cytoplasmic gp210-GFP co-localized 92

extensively with VAPB in tubular membranes. Its NE localization was unaffected by up to 3-fold 93

overexpression of the transgene based on fluorescence intensity measurements relative to 94

endogenous VAPB. In contrast, gp210-GFP was excluded from the NE and retained in mutant 95

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VAPB-containing dilated cytoplasmic membranes (Fig. 2A). Similarly, Nup214-GFP that 96

normally localizes to the NE was sequestered in mutant VAPB-containing cytoplasmic 97

membranes, albeit not all mutant VAPB-containing membranes were Nup214-GFP positive (Fig. 98

2B). This effect was not cell line specific as HeLa showed similar Nup214 relocation defect 99

(Supplemental Fig. S1). The normal NE localization of both gp210-GFP and Nup214-GFP were 100

restored upon co-overexpression of the FFAT fragment (Fig. 2A,B), consistent with the ability of 101

this motif to resolve mutant VAPB effects. Thus, cytoplasmic retention of these Nups along with 102

aggregated mutant VAPB inhibits their transport to the NE. 103

To determine whether trafficking of other NE proteins is also affected, we examined the 104

distribution of EMD, an integral membrane protein that shuttles between the ER and INM 105

(Zuleger et al., 2011). Endogenous EMD formed a ring-like pattern along the rim of the NE in 106

both control and VAPB-WT transfected cells (Fig. 2C). It partially co-localized with VAPB-WT 107

in cytoplasmic puncta throughout the cell as well as in discreet regions adjacent to or at the NE. 108

In contrast, EMD was retained in dilated cytoplasmic membranes and excluded from the NE in 109

mutant VAPB overexpressing cells (Fig. 2C). These EMD-containing membranes were often 110

positioned adjacent to but not part of the NE. Co-overexpression of the FFAT fragment with 111

VAPB-WT had no effect on EMD distribution, but restored EMD localization to the NE in 112

mutant VAPB cells (Fig. 2C). Since EMD is retained in the INM in part by binding to A-type 113

lamins in the nuclear lamina (Vaughan et al., 2001; Ostlund et al., 2006), we examined the 114

distribution of Lamin A/C and found no change in its distribution pattern or evidence of nuclear 115

deformation (J.K.N., unpublished data). Thus, loss of EMD from the INM is likely due to 116

disruption of transport to the INM rather than loss of retention at the INM. 117

Cytoplasmic Retention is due to Loss of VAPB Function 118

Mutant VAPB is aggregation-prone and recruitment of endogenous VAPB to insoluble 119

aggregates is thought to result in a dominant negative effect (Teuling et al., 2007; Suzuki et al., 120

2009; Kim et al., 2010). To determine whether the defect is due to loss of VAPB function and to 121

exclude non-specific sequestration of NE proteins with aggregated mutant VAPB, we examined 122

their distribution upon siRNA knockdown of endogenous VAPB. Co-transfection with the empty 123

pLKO.1 vector had no effect on the distribution pattern of Nup214-GFP, whereas siVAPB 124

resulted in relocation of Nup214-GFP to cytoplasmic foci and loss of NE localization (Fig. 3A). 125

The cytoplasmic foci likely represent sites of NPC assembly since Nup214 is a soluble Nup 126

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recruited to the membrane to form the core scaffold during NPC assembly (Bodoor et al., 1999). 127

This accumulation of Nup214-GFP suggests that VAPB is required for transport of pre-128

assembled NPC to the NE. It is consistent with the view that mutant VAPB acts in a dominant 129

negative manner. 130

Transport of EMD was similarly inhibited by knockdown of VAPB. Endogenous EMD 131

was relocated from the NE to large cytoplasmic puncta throughout the cell upon siVAPB (Fig. 132

3B). Thus, VAPB function is essential for transport of Nup214, EMD and possibly other 133

membrane proteins to the NE. Loss of NE localization is not simply due to inadvertent 134

sequestration of Nups with mutant VAPB aggregates but a consequence of loss of VAPB 135

function. 136

VAPB Affects ERGIC Trafficking 137

To identify this VAPB-containing intracellular compartment, we examined its co-138

localization with known organelle markers. Only cells with low level of expression of transfected 139

VAPB-WT were examined to avoid potential overexpression artifacts. We found VAPB-WT co-140

localized extensively with GFP-ERGIC-53 cytoplasmic puncta (Fig. 4A). The perinuclear Golgi 141

ribbons were devoid of VAPB, suggesting VAPB resides primarily at ERGIC. When subjected 142

to intensity correlation analysis, excluding the Golgi ribbons, VAPB and ERGIC-53 showed a 143

mean Pearson’s correlation and Mander’s overlap coefficients of 0.73 ± 0.01 and 0.79 ± 0.02 144

(from 3 replicates of 10-17 cells), respectively. These high coefficients suggest co-localization 145

with recycling ERGIC-53. To determine whether VAPB affects trafficking through ERGIC, we 146

examined the distribution of endogenous ERGIC-53 upon siVAPB knockdown. ERGIC-53 147

relocated from the Golgi to expanded membranes in 45.6% ± 0.9% (n=3) of siVAPB cells (Fig. 148

4B) compared to 4.9% ± 1.9% in pLKO.1 control, indicating that relocation was not an artifact 149

of ectopic ERGIC-53 expression. Quantitation of individual peripheral cytoplasmic puncta 150

indicated a shift towards larger ERGIC-53 puncta in the knockdown cells (Fig. 4C). This 151

indicates that VAPB is essential in maintaining ERGIC morphology and retrograde trafficking of 152

ERGIC-53. To verify that Nups are retained in ERGIC in a VAPB-dependent manner, we stained 153

the knockdown cells with mAb414, a monoclonal anybody that recognizes several FG repeat 154

Nups (Davis and Blobel, 1986). These Nups were normally localized to the rim of the NE and in 155

small cytoplasmic puncta in control cells, but were retained in expanded GFP-ERGIC-53-156

containing membranes upon siVAPB (Fig. 4C). Distribution of EMD was similarly affected, 157

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relocating to the expanded ERGIC along with GFP-ERGIC-53 in siVAPB cells (Fig. 4D). 158

Quantitation of mAb414 and EMD cytoplasmic puncta excluding the NE also indicated a shift 159

towards larger puncta in the knockdown cells (Fig. 4C). Together, this indicates that NE and 160

transmembrane Nups transit through ERGIC, and VAPB is required for final transport to the NE. 161

Our study shows that overexpression of mutant VAPB not only causes ER abnormalities 162

but also separation of the ONM from the INM at discreet regions of the NE. Interestingly, this 163

abnormality is similar to that caused by disruption of gp210 (Drummond and Wilson, 2002), 164

which leads to formation of nuclear pore intermediates that fail to dilate into functional pores. 165

This suggests that positioning of fully assembled pores at the junction of the two nuclear 166

membranes structurally contributes to their close apposition. Cytoplasmic retention of certain 167

Nups and membrane proteins upon siRNA knockdown clearly indicates that VAPB plays an 168

essential role in their transport to the NE. This disruption in transport will likely affect the 169

overall composition of NPC and compromise the structural integrity as well as functional 170

properties of the pores. Interestingly, bulk anterograde ER-to-Golgi transport of VSVG remains 171

largely unaffected upon siVAPB knockdown (Fig. S2), suggesting that VAPB is primarily 172

involved in regulating retrograde transport through ERGIC. 173

Co-overexpression of the FFAT fragment clearly counteracts the adverse effects of 174

mutant VAPB. We propose that interaction with the FFAT fragment might interfere with mutant 175

VAPB aggregate formation. The P56S mutation does not directly affect FFAT binding nor does 176

it result in complete loss of function since the mutant protein is able to rescue the Drosophila 177

deletion phenotype (Chai et al., 2008). Its susceptibility to form insoluble aggregates may recruit 178

endogenous VAPB (Suzuki et al., 2009), and block access to the FFAT binding site as the 179

aggregates increase in size (Kim et al., 2010). This not only generates a dominant negative effect 180

but possibly additional toxic properties. Binding to the FFAT fragment may impart 181

conformational changes that reduce aggregate formation, allowing partial restoration of wild-182

type functions and averting aggregate toxicity. 183

Cytoplasmic retention of Nups and EMD in mutant VAPB-containing membranes is not 184

due to inadvertent sequestration with insoluble aggregates since siRNA knockdown of 185

endogenous VAPB also results in their cytoplasmic retention. Given that wild-type VAPB co-186

localizes with recycling ERGIC-53, and siVAPB knockdown results in expansion and retention 187

of Nups and EMD with ERGIC-53, it suggests that these NE membrane proteins do not reach the 188

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NE by lateral diffusion through the interconnecting ER and ONM network, but transit through 189

ERGIC. Progression from these ERGIC foci is clearly dependent on VAPB with its loss of 190

function either through siRNA knockdown or dominant negative effect of mutant VAPB 191

overexpression inhibiting their exit and consequently expansion of ERGIC. While the 192

mechanism by which VAPB facilitates this retrograde transport step remains to be determined, 193

progressive deterioration of the NE is a predictable consequence of disrupting this transport step 194

and could contribute to age-dependent onset of the disease. 195

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Materials and Methods 197

Expression Plasmids, Cell Culture and Transfection 198

FLAG-tagged VAPB and Myc-tagged FFAT constructs were described previously 199

(Prosser et al., 2008). Gp210-GFP and Nup214-GFP were from EUROSCARF. Chinese Hamster 200

Ovary (CHO-K1) and HeLa were maintained at 37°C in MEMα and DMEM (Invitrogen, 201

Carlsbad, CA, USA), respectively, and supplemented with 100 units/ml penicillin, 100 µg/ml 202

streptomycin and 10% fetal bovine serum (FBS). Cells were transfected with LipofectAMINE 203

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 204

Immunofluorescence and Electron Microscopy 205

Transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline 206

(PBS) for 30 min at room temperature. After neutralization with PBS-glycine (PBS with 100 207

mM glycine), cells were permeabilized in blocking buffer (PBS, 1% bovine serum albumin, 2% 208

normal goat serum, 0.4% saponin and 0.1% Triton X-100). Primary and secondary antibodies 209

were diluted in blocking buffer and incubated for at least 1h. Cover slips were mounted with 210

SlowFade Gold with or without DAPI (Invitrogen Carlsbad, CA, USA). Primary antibodies used 211

included anti-FLAG (Applied Biological Materials, Richmond, BC, Canada), mAb414 212

(Covance, Princeton, NJ, USA), ERGIC-53 (Sigma-Aldrich, St. Louis, MO, USA), and EMD 213

(MANEM1, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Secondary 214

antibodies were conjugated with Alexa 488 or 594 (Invitrogen, Carlsbad, CA, USA). 215

Images were captured on a LSM 510confocal microscope with a 1.4 numerical aperture 216

63X oil-immersion objective and processed with Image J (NIH, Bethesda, MD, USA). ERGIC-217

53, mAb414 and EMD cytoplasmic particles sizes excluding the perinuclear Golgi ribbons and 218

NE were measured with particle analysis plug-in. Over 10 cells were chosen from each group 219

and from 3 replicates. Maximal Feret’s diameter of individual peripheral puncta (>200 per cell) 220

was grouped into 100 nm bins. Unpaired two-tailed Student’s t-test was used to determine 221

statistical significance. 222

For electron microscopy, cells were fixed with 1.6% glutaraldehyde in 0.1 M sodium 223

cacodylate buffer pH 7.2 for 2-6 h. After contrasting with 1% osmium tetroxide and dehydration 224

in increasing concentrations of ethanol, cells were embedded in Spurr’s low viscosity epoxy 225

(Polysciences, Warrington, PA, USA). Resulting ultra-thin sections were stained with 5% Uranyl 226

Acetate for 15 min and Reynold’s Lead Citrate Solution for 5 min. Digital images were taken 227

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with a JEOL 1230 transmission electron microscope at 60kV adapted with a 2000 x 2000 pixel 228

bottom mount Hamamatsu CCD digital camera. 229

Lentiviral pLKO.1 based plasmids were from Open Biosystems (Hunstville, AL, USA). 230

TRCN0000153862 and TRCN0000152888 matched both human and mouse VAPB sequences. 231

Empty pLKO.1 vector or eGFP shRNA were used as controls. In some cases, co-transfection 232

with monomeric RFP (mRFP) was used to identify the transfected cells. Cells were processed 233

48h post-transfection. 234

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Acknowledgements 236

This work was supported by an operating grant from the Canadian Institutes of Health Research. 237

A.C. was supported by NSERC of Canada. We thank Ms. Kalina Abrol for her technical support. 238

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Figure Legends 240

Figure 1. Overexpression of VAPB-P56S causes NE defect. (A) Large vacuole-like membrane 241

structures (*) and swelling of nuclear envelope (arrows) in mutant VAPB transfected CHO cells 242

after 48h. (B) Magnified image of the NE defect with some containing membrane vesicles within 243

the perinuclear space (arrowhead). (C) Magnified image of a dilated interconnecting ER tubule 244

(#). Scale bar, 500 nm. (D) Measurement of the gap between ONM and INM in control and 245

mutant VAPB transfected cells. 246

Figure 2. NE proteins are retained in mutant VAPB-containing membranes. CHO cells were co-247

transfected with FLAG-tagged VAPB (WT or P56S) and gp210-GFP (A) or Nup214-GFP (B). 248

FFAT was co-transfected with mutant VAPB in the bottom panels. (C) Cells were transfected 249

with FLAG-VAPB-WT or P56S and stained with anti-FLAG (red) and anti-EMD (green). FFAT 250

was co-transfected with mutant VAPB in the bottom panel. Scale bar, 10 µm. All images are 251

representative of at least 90% of transfected cells from three replicates. 252

Figure 3. Cytoplasmic retention of Nup214-GFP and EMD upon siVAPB knockdown. (A) HeLa 253

was co-transfected with Nup214-GFP and Lentiviral constructs (empty pLKO.1 or siVAPB 254

152888) and mounted with DAPI. (B) Endogenous EMD distribution in cells co-transfected with 255

mRFP and empty pLKO.1 or siVAPB. Scale bar, 10 µm. 256

Figure 4. VAPB is localized to the ERGIC. (A) HeLa was transfected with VAPB-WT and GFP-257

ERGIC-53. Only low expressing cells were analyzed. (B) Distribution of endogenous ERGIC-53 258

in cells co-transfected with empty pLKO.1 or siVAPB and mRFP. (C) Distribution histograms of 259

maximal Feret’s diameter of the cytoplasmic puncta in 100 nm bins for ERGIC-53, mAb414 and 260

EMD. Asterisks (*) indicate p<0.05. (D) Cells were transfected as in (B), but further stained with 261

mAb414 antibodies or with anti-EMD antibodies (E). Scale bar, 10 µm. 262

Supplemental Figures 263

Figure S1. Cellular distribution of Nup214 in HeLa. HeLa cells were co-transfected with FLAG-264

VAPB (WT or P56S), and Nup214-GFP. Cells were fixed after 48h and stained with anti-FLAG 265

antibodies. Scale bar, 10 µm. 266

Figure S2. Transport of anterograde VSVGts042-GFP in siVAPB knockdown cells. (A) HeLa cells 267

were co-transfected with pLKO.1 empty vector or siVAPB and VSVGts042-GFP. VSVGts042-GFP 268

was trapped in the ER at 42ºC for 5h, and shifted to 32ºC for the time indicated before fixation. 269

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(B) Fraction of cells with Golgi-localized VSVGts042-GFP after release from 42ºC. Error bars 270

represent s.e.m. (n=3). 271

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