Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment
- Marijn Kuijpers1, 2Email author,
- Vera van Dis1Email author,
- Elize D Haasdijk1,
- Martin Harterink2,
- Karin Vocking3,
- Jan A Post3,
- Wiep Scheper4, 5,
- Casper C Hoogenraad1, 2Email author and
- Dick Jaarsma1Email author
© Kuijpers et al.; licensee BioMed Central Ltd. 2013
Received: 31 May 2013
Accepted: 1 June 2013
Published: 12 June 2013
Protein aggregation and the formation of intracellular inclusions are a central feature of many neurodegenerative disorders, but precise knowledge about their pathogenic role is lacking in most instances. Here we have characterized inclusions formed in transgenic mice carrying the P56S mutant form of VAPB that causes various motor neuron syndromes including ALS8.
Inclusions in motor neurons of VAPB-P56S transgenic mice are characterized by the presence of smooth ER-like tubular profiles, and are immunoreactive for factors that operate in the ER associated degradation (ERAD) pathway, including p97/VCP, Derlin-1, and the ER membrane chaperone BAP31. The presence of these inclusions does not correlate with signs of axonal and neuronal degeneration, and axotomy leads to their gradual disappearance, indicating that they represent reversible structures. Inhibition of the proteasome and knockdown of the ER membrane chaperone BAP31 increased the size of mutant VAPB inclusions in primary neuron cultures, while knockdown of TEB4, an ERAD ubiquitin-protein ligase, reduced their size. Mutant VAPB did not codistribute with mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, and accumulate in a protective ER derived compartment termed ERPO (ER protective organelle) in neurons.
The data indicate that the VAPB-P56S inclusions represent a novel reversible ER quality control compartment that is formed when the amount of mutant VAPB exceeds the capacity of the ERAD pathway and that isolates misfolded and aggregated VAPB from the rest of the ER. The presence of this quality control compartment reveals an additional level of flexibility of neurons to cope with misfolded protein stress in the ER.
KeywordsAmyotrophic lateral sclerosis (ALS) Protein aggregation ER associated degradation Motor neuron disease Mouse model
Protein aggregation is a central feature of many neurodegenerative disorders, including Alzheimer's disease, Parkinson’s disease and amyotrophic lateral sclerosis (ALS). Aggregation-prone proteins may accumulate into discrete micrometer-scale structures that are termed inclusions, inclusion bodies, aggregates or have disease or morphology specific names (e.g. Lewy bodies, Pick bodies, neurofibrillary tangles), and can be correlated to specific disorders . Not only the protein composition, but also the morphologies as well as (sub) cellular and regional distributions of inclusions can be correlated to specific disorders and subtypes of disorders [1–3]. Depending on the type of disorder and inclusion, inclusions may be either neuroprotective, neutral or detrimental structures, and precise knowledge about their characteristics is instrumental for our understanding of neurodegenerative disorders [1, 4].
A peculiar inclusion that is ultrastructurally characterized by the presence of ER-derived membranous profiles occurs in cellular and invertebrate models of a familial ALS-like disorder designated ALS8 [5–7]. ALS8 is caused by mutation in VAPB , a small tail-anchored ER membrane protein that is member of a conserved VAP (VAMP/synaptobrevin-associated proteins) family of proteins. Several VAPB mutations have been identified, but so far only a P56S mutation is yet known to co-segregate with disease [8, 9]. VAP proteins are characterized by an N-terminal MSP (major sperm protein) domain, a coiled-coil motif, and a C-terminal transmembrane region, and in mammals consists of two genes, VAPA and VAPB[10, 11]. The MSP domain (named after C. elegans MSP) contains a binding site for the FFAT (diphenylalanine [FF] in an acidic tract) that are present in a variety of proteins [12, 13]. In addition, the MSP domain may function as a secreted ligand after cleavage from the transmembrane domain . VAPs have been implicated in multiple function including non-vesicular transfer of lipids and membrane trafficking, ER-organelle and ER-cytoskeleton interaction and homeostatic and signaling functions at the neuromuscular synapse [10, 14–16].
The P56S mutation causes rapid oligomerization and aggregation of mutant VAPB, and typically accumulates in multiple dot-like inclusions in transfected cells and animal models, including transgenic mice [6, 8, 17, 18]. Several mechanisms by which mutant VAPB causes ALS have been proposed, including a dominant negative mode of action by recruiting wild-type VAPB and VAPA or other factors into aggregates, a gained toxic activity, or partial loss of function [5, 6, 15, 19–21]. The aim of this study was to further characterize mutant VAPB inclusions in vivo in neurons of P56S-mutant VAPB transgenic mice. The data indicate that mutant VAPB inclusions that occur in motor neurons of these mice represent a specialized ER associated protein quality control compartment that isolates misfolded and aggregated VAPB targeted for degradation from the rest of the ER. The presence of this quality control compartment in addition to the ER associated degradation machinery may explain the late onset of mutant VAPB-induced disease in man.
Animals were housed and handled in accordance with the “Principles of laboratory animal care” (NIH publication No. 86–23) and the guidelines approved by the Erasmus University animal care committee.
A selected group of different transgenic lines was allowed to age for 2 years (Additional file 1: Table S1). These mice were weighed and inspected for signs of muscle weakness once a week, using a set of simple tests: i.e. the mice were examined for their ability to extend their hindlimbs when suspended in the air by their tail, and their ability to hang upside down on a grid for 60 s . In addition, at specific ages animals were subjected to an accelerating rotarod test as described . The mice were killed when they developed motor problems or when they reached 2 years of age (Additional file: 1 Table S1). A subset of mice was excluded from the study because of non-motor related discomfort (e.g. eye infections or tumors; see Additional file 1: Table S1). Selected mice were analyzed for neuromuscular denervation and pathological abnormalities in the spinal cord (e.g. motor neuron loss, gliosis).
Axotomy of the sciatic nerve
Six weeks old hemizygote hVAPB-P56S mice from the VM1 line and their non-transgenic littermates were anesthetized. The left sciatic nerve was exposed, bound with suture and cut at mid-thigh level. After various intervals mice were perfused transcardially with 4% paraformaldehyde and processed for immunocytochemistry.
Primary antibodies reported in this study are: mouse anti-actin (Millipore); mouse anti-αB-crystallin (Stressgen Biotechnologies); rabbit anti-ATF3 (Santa Cruz Biotechnology); rabbit anti-BAP31 rabbit (gift from M. Tagaya; Tokyo University of Pharmacy and Life Sciences ); rabbit anti-BiP/GRP78 (Stressgen Biotechnologies); rabbit anti-calreticulin (Affinity BioReagents); mouse anti-CD8 (SantaCruz); goat-anti-choline acetyltransferase (ChAT, Millipore); rabbit-anti-CGRP (Calbiochem); rabbit anti-Derlin-1 (D4443, Sigma-Aldrich); rabbit anti-GFAP (DAKO); mouse-anti GM130 (BD Biosciences); mouse anti-HA (Covance); rat anti-HA (Roche); rabbit anti-HA (Santa Cruz Biotechnology); rabbit anti-Iba1 (WAKO Chemicals); rat anti-Mac2 (Cedarlane); rabbit anti KDEL (Stressgen Biotechnologies); rabbit anti-myc (Cell Signaling Technology); mouse anti-myc (Santa Cruz Biotechnology); mouse anti-NeuN (MAB377, Millipore); chicken anti-neurofilament M (Millipore), rabbit anti-NIR2 (Santa Cruz Biotechnology); rabbit anti-ORP9 (gift from Neale Ridgway, Dalhousie University, Canada); rabbit anti-ORP2, rabbit anti-ORP3, rabbit anti-ORP6 (gifts from Vesa Olkkonen, Institute for Molecular Medicine Finland); human anti-ribosomal protein P0 (Immunovision); Rabbit anti-phosphoS6 (Cell Signaling Technology), mouse anti-ubiquitin (FK2; 1:300; Biomol); rabbit anti-VAPB ; guinea pig anti-VAChT (Millipore); mouse anti-VCP (Ma3-004; Thermo Scientific).
Secondary antibodies: For avidin-biotin-peroxidase immunocytochemistry biotinylated secondary antibodies from Vector Laboratories diluted 1:200 were used. FITC-, Cy3-, and Cy5-conjugated secondary antibodies raised in donkey (Jackson Immunoresearch, USA), Alexa488, 568 or 633 conjugated antibodies raised in goat were used for immunofluorescence. For Western blots, HRP-conjugated goat-anti mouse or goat-anti rabbit IgG were used at 1:5000 (DAKO, 1:5000).
Tissue samples were homogenized in ten volumes of PBS containing 0.5% Nonidet P-40 and 1× protease inhibitor cocktail (Complete, Roche), sonicated and centrifuged at 800 g for 15 min at 4°C to obtain the S1-supernatant. For the preparation of detergent-insoluble extracts, S1 supernatants were centrifuged at 15000 g for 20 min. After the collection of supernatants (S2), pellets (P2) were thoroughly washed five times with PBS-0.5% Nonidet P-40 and then resuspended in sample buffer for SDS–PAGE electrophoresis and western blotting. Protein concentrations in samoles were determined using the BCA method (Pierce, Rockford, IL).Samples containing 5–50 mg protein were electrophoresed on SDS–PAGE gels and blotted on PVDF membranes (Millipore). The membranes were blocked with 5% non-fat dry milk (Bio-Rad) in PBS with 0.05% Tween20 (PBST), incubated in primary antibody, diluted in PBST with 1% dry milk followed by incubation in secondary antibody. Blots were exposed to film after incubation in chemiluminescence’s reagent (ECL, Amersham), and films were analyzed with Metamorph software.
RT-PCR of unfolded protein responsive genes
Levels of unfolded protein stress responsive mRNAs were analyzed by real time quantitative reverse transcription PCR (qRT-PCR) using the Roche LightCycler 480 and the Roche universal probe library as described . RNA was isolated from cortex samples using Trizol reagent and used for cDNA synthesis. Primers for the qRT-PCR assay were: BiP FW: gccaactgtaacaatcaaggtct/RV: tgacttcaatctggggaactc (probe #15) and Chop FW: ccaccacacctgaaagcag/RV: tcctcataccaggcttcca (probe #33). Values are normalized to eEF2α mRNA FW: acacgtagattccggcaagt/RV: aggagccctttcccatctc (probe #31) for individual animals .
Primary neuron cultures and transfection
Primary hippocampal cultures were prepared from embryonic day 18 (E18) rat brains . Cells were plated on coverslips coated with poly-L- lysine (30 μg/ml) and laminin (2 μg/ml) at a density of 75,000/well. Hippocampal cultures were grown in Neurobasal medium (NB) supplemented with B27, 0.5 mM glutamine, 12.5 μM glutamate and penicillin/streptomycin. Hippocampal neurons were transfected using Lipofectamine 2000 (Invitrogen). The following mammalian expression plasmids have been described previously: HA- and myc-tagged VAPB-wt and VAPB-P56S constructs ; myc-tagged seipin-wt, seipin-N88S and seipin-S90L constructs ; and BAP31-mRFP construct . ΔTM-VAPB-P56S-GFP was generated by a PCR-based strategy using HA-VAPB-P56S construct as a template and subcloned into a GFP-tagged pβactin expression vector. HA-VAPB-P56S-CD8TM was made by removing the transmembrane domain of VAPB-P56S and adding the transmembrane domain of CD8 with a PCR-based strategy using HA-VAPB-P56S and GFP-CD8  as a template and subcloned into a pβactin expression vector. BAP31 (5’-gagaatgatcagctaaaga-3’) and TEB4 (5’-ttaagagcctcttgcctca-3’) shRNA construct sequences were designed based on previously published sequences [29, 30]. The complementary oligonucleotides were annealed and inserted into a pSuper vector . DNA (3.6 μg /well) was mixed with 3 μl of Lipofectamine 2000 in 200 μl of medium, incubated for 30 min, and then added to the neurons in NB at 37°C in 5% CO2 for 45 min. Next, neurons were washed with NB and transferred in the original medium at 37°C in 5% CO2. 2–4 days after transfection, neurons were fixed with 4% paraformaldehyde/4% sucrose in PBS, washed three times in PBS for 10 min and incubated with the indicated primary antibodies in GDB buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4) overnight at 4°C. Following incubation with secondary antibody neurons were mounted using Vectashield mounting medium (Vector laboratories). Images for co-localization measurements were acquired using a Nikon microscope equipped with a 100x oil objectives. Confocal images were acquired using a LSM510 confocal microscope (Zeiss) with 40x or 63x oil objectives.
Immunohistochemical and histopathological procedures
For immunocytochemistry and immunofluorescence mice were anaesthetized with pentobarbital and perfused transcardially with 4% paraformaldehyde. The lumbar and cervical spinal cord were carefully dissected out and post-fixed overnight in 4% paraformaldehyde. Routinely, spinal cord tissue was embedded in gelatin blocks, sectioned at 40 μm with a freezing microtome and sections were processed, free floating, employing a standard avidin-biotin-immunoperoxidase complex method (ABC, Vector Laboratories, USA) with diaminobenzidine (0.05%) as the chromogen, or single, double and triple-labelling immunofluorescence . Immunoperoxidase-stained sections were analyzed and photographed using a Leica DM-RB microscope and a Leica DC300 digital camera. Sections stained for immunofluorescence were mounted on coverslips, placed on glass slides with Vectashield mounting medium, and were examined with Zeiss LSM 510 and LSM 700 confocal laser scanning microscopes.
For analysis of neuromuscular denervation medial gastrocnemius muscle from 4% paraformaldehyde fixed mice were dissected, embedded into gelatin blocks and sectioned at 80 μm with a freezing microtome . Sections were immunolabeled, free floating, for guinea pig anti-VAChT and chicken-anti-NFM followed by Cy3 anti-goat and Cy5 anti-chicken or anti-rabbit secondary antibody, and motor endplates were labeled with FITC-bungarotoxin (1:500, Molecular Probes). For quantitative analyses, muscle sections were examined under a Leica DM-RB fluorescence microscope, end-plates being scored as ‘innervated’ in case of complete overlap between bungarotoxin and VAChT labeling, ‘partially denervated’ in case of partial overlap, and ‘denervated’ in case of the absence of VAChT labeling at the end-plate.
Quantitative analysis of immunofluorescence images
Fluorescent intensities and inclusion sizes were determined using Metamorph image analysis software. Images were collected using Zeiss LSM 510 confocal laser scanning microscope with 63x Plan apo oil immersion objective. Analyses of inclusions in cultured neurons were performed on maximal projections of confocal stacks. For analysis of FK2-labeled motor neurons stacks of 1 μm thick sections were collected from the first 4 μm facing the coverslip, and the optical section 2 μm below the surface of the section was used for density measurements. Material from non-transgenic and transgenic mice always was imbedded in a single gelatin block to minimalize variability in staining intensity resulting from the sectioning and immunostaining procedure . Per mouse, motor neurons from 3 randomly selected L4 sections (yielding 4–12 cells/sections) were measured.
Analyses of sciatic nerves
Sciatic nerves were carefully dissected from perfused mice, post-fixed in 4% paraformaldehyde with 1% glutaraldehyde, extensively rinsed in 0.1 M PB, post-fixed in 1% osmium, dehydrated, embedded in Durcupan, sectioned transversely at 0.5 μm with an Ultratome, and stained with toluidine blue.
Transmission electron microscopy
For electron microscopy mice were perfused transcardially with 4% paraformaldehyde with 0.2% (post-embedding immunogold electron microscopy) or 1% (standard transmission electron microscopy) glutaraldehyde. Specimens were sectioned with a Vibratome and further processed using standard methods as described before [22, 32]. For standard transmission electron microscopy Vibratome section (60–100 μm) were post-fixed in 1% osmium, dehydrated and embedded in Durcupan. Ultrathin sections (50–70 nm) were contrasted with uranyl acetate and lead citrate, and analyzed in a Phillips CM100 electron microscope at 60 or 80 kV.
Post-embedding immunogold labeling was performed on 50–70 nm thick thin sections from 4% paraformaldefyde and 0.2% glutaraldehyde fixed brain and spinal cord sections as described before  using the rat-anti-HA antibody at 1:100.
For electron microscopic analysis of VAPB-P56S inclusions in HeLa cells, cells were transfected with Myc-hVAPB-P56S and the Addgene plasmid 40307 to enable selection of transfected cells under the electron microscope . 24 h after transfection cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer, stained for DAB , post-fixed with 1% OsO4 (EMS) and 1.5% K4[Fe(CN)6] in cacodylate buffer (90 min on ice), followed by 1% low molecular weight tannic acid (30 min at RT) and with 1% OsO4 in distilled water (30 min on ice) as described , embedded in epon, sectioned at 50 nm, and contrasted with uranyl acetate .
Statistical analyzes were performed with MS Excel or Graphpad Prism software (San Diego, USA). Means from different age groups, and different transgenic mouse lines were compared using Student’s t-test, or one-way ANOVA and Tukey’s post-test.
Mutant VAPB inclusions are positive for luminal ER proteins and are surrounded by ribosome-rich areas
To determine whether the presence of inclusions was associated with altered solubility of mutant VAPB we performed Western blot analysis of non-ionic detergent (Nonidet P40)-insoluble (P2) fraction of spinal cord homogenate. In accordance with reduced solubility a large proportion of transgenic mutant VAPB accumulated in the insoluble fraction (Additional file 1: Figure S2). Endogenous murine VAPB was not detectable in this fraction, suggesting that it does not coaggregate with transgenic mutant VAPB (Additional file 1: Figure S2).
To characterize the mutant VAPB inclusions we first double stained for HA or VAPB and a variety of cellular markers. Double labeling with antibodies against calreticulin (a luminal ER sugar-binding protein) and KDEL (a C-terminal tetrapeptide motif shared by several ER chaperones), showed that the mutant VAPB inclusions were immunoreactive for these ER markers (Figure 2C-F). However calreticulin and KDEL staining was not enriched in the inclusions; the same staining intensity is observed in the inclusions as compared to the surrounding area. Accordingly, the calreticulin and KDEL staining in motor neurons with inclusions was indistinguishable from that in non-transgenic motor neurons (e.g. compare Figure 2C and 2E with 2D’ and F’, respectively). Double labeling with antibodies against ribosomal protein P0 and phosphorylated ribosomal protein S6 showed that while the inclusions were immunonegative for P0 and phospho-S6, the area around the inclusions contains a high density of ribosomes (Figure 2G, H). The specific association of mutant VAPB inclusions within ribosome-rich areas was particularly clear in motor neuronal dendrites, which showed areas of intense phospho-S6 staining. Analysis of a large number of dendritic VAPB inclusions (> 100) indicated that in all occasions they were present within an area of intense phospho-S6 staining (Figure 2H’).
Mutant VAPB did not codistribute with the Golgi apparatus marker GM130, and the presence of inclusions did not have a detectable effect on the Golgi apparatus morphology (Figure 2I, J). Also markers for lysosomes (LAMP1, Figure 2K) and endosomes (EAA1, not shown) did not codistribute with inclusions and showed unaltered distributions in motor neurons with inclusions. Finally we screened antibodies against a variety of FFAT-motif containing proteins, representing a major class of VAPB interacting proteins [6, 37, 38] to determine whether these proteins accumulate in the mutant VAPB inclusions. Antibodies against NIR2 and ORP9 produced consistent labeling in motor neurons. However, ORP9 (Figure 2L) nor NIR2 (data not shown) immunoreactivity was present in the mutant VAPB inclusions, consistent with the observation that the P56S mutation disrupts the FFAT-motif binding domain of VAPB .
VAPB inclusions ultrastructurally are characterized by smooth ER-like profiles and electron dense material
Summary of ER abnormalities in Thy1.2-hVAPB transgenic lines
Electron dense stacked ER
VM1 + VM5
Stacked ER cisterns in wt-hVAPB motor neurons
Gradual loss of mutant VAPB inclusions in axotomized motor neurons
To further investigate the connection between ATF3 expression, axonal damage and the absence of VAPB inclusions, we examined the effect of axotomy on inclusions in sciatic nerve motor neurons of VM1 mice. Axotomy results in a strong induction of ATF3 expression in motor neurons within 12 hours, which lasts for more than 5 weeks post transection [44, 46]. Analysis of inclusions in axotomized ATF3-positive motor neurons revealed a gradual reduction of the size of inclusions starting within 24 hours post-axotomy (Figure 5D-G). Ultimately, axotomy resulted in the total absence of inclusions and diffuse HA labeling 2–3 weeks post-axotomy (Figure 5D-G). The diffuse HA labeling in 2–3 weeks axotomized VM1 motor neurons strongly resembled HA-labeling in motor neurons of MP1 and MP2 mice described above. Importantly, the level of HA-labeling in 2–3 weeks axotomized VM1 motor neurons is considerable higher than in VM2 motor neurons, indicating that the absence of inclusions cannot simply be explained by reduced VAPB-P56S expression levels. Together the data indicate that mutant VAPB inclusions in motor neurons of VAPB-P56S transgenic mice are reversible ER-associated structures.
VAPB inclusions are immunoreactive for proteins of the ER associated degradation (ERAD) quality control pathway
We next stained for Valosin-containing protein (VCP/p97, cdc48 in yeast), which is an essential ERAD component that provides mechanical force for extracting substrate from the ER membrane . In wild-type motor neurons VCP-immunoreactivity was present in the nucleus and the perikaryon, with higher staining intensities of the nucleus (Figure 6I). Motor neurons with mutant VAPB inclusions showed the same overall staining, but in addition showed intense VCP staining within the inclusions, indicative of an accumulation of VCP in the inclusions (Figure 6E). Staining for Derlin-1, an ER membrane spanning protein that plays a role in ERAD of many substrates, including tail-anchored proteins , showed that VAPB inclusions were also immunoreactive for Derlin-1 (Figure 6F). However, unlike VCP, Derlin-1 immunoreactivity was not specifically enriched in the inclusions, showing the same overall distribution in non-transgenic motor neurons and motor neurons with inclusions (compare Figure 6F’ and H). Finally, we stained for BAP31 an ER membrane chaperone that may play a role in ERAD [24, 51], and is enriched in mutant VAPB inclusions in transfected HeLa cells . Accordingly, we found a marked enrichment of BAP31 immunoreactivity in mutant VAPB inclusions in our transgenic mice (Figure 6G). Together the data suggest that mutant VAPB inclusions are a region of increased ERAD activity.
VAPB inclusions may represent an ER associated degradation (ERAD) quality control compartment
VAPB-P56S inclusions differ from an ER protective organelle (ERPO) associated with luminal ERAD substrates
Inclusions in motor neurons of transgenic VAPB-P56S mice may represent an ER quality control compartment
In the present study we show that transgenic mice expressing an ALS8-linked mutant form of VAPB develop a novel type of inclusion that is associated with the rough ER and consists of smooth ER-like tubular profiles and electron dense material. Several lines of evidence indicate that this mutant VAPB inclusion represents an ER protein quality control compartment: First, the presence of the inclusions was not associated with signs of neuronal malfunction or neuronal pathology. Second, the inclusions are localized in the center of healthy appearing active rough ER. Third, the inclusions are reversible as they gradually disappear following axonal transection. Fourth, the inclusions are enriched in factors that operate in the ER associated degradation (ERAD) pathway, i.e. p97/VCP, Derlin-1 and the ER membrane chaperone BAP31. And fifth, inhibition of ERAD increased the size of the inclusions. We propose that the inclusions in our mutant VAPB transgenic mice represent an ER quality control compartment that arises, when the amount of substrate exceeds the capacity of ER associated degradation (ERAD). This ERAD associated quality control compartment is reminiscent of aggresomes that may form in condition of excess of cytosolic aggregation-prone protein [4, 56]. Compatible with this idea, mutant VAPB inclusions do not occur in motor neurons of low-expressing mutant VAPB transgenic mice (line VM2), and are absent in motor neurons derived from induced pluripotent stem cells of ALS8 patients .
Several studies have reported on ER-derived inclusion-like structures that are formed after expression of mutated ER proteins in yeast or mammalian cells [55, 58–60]. An ER-derived structure termed ERPO (ER protective organelle) has been identified as a protective ER compartment in cells expressing the serpin α1-antitrypsin with an E342K mutation, associated with liver disease in children . ERPO may represent an ER quality control pathway for multiple ER substrates, since aggregation-prone mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, accumulate in the same compartment . Our data show that mutant VAPB and mutant seipin accumulate in different inclusions when coexpressed, indicating that the mechanisms that operate in the formation of VAPB inclusions differ from those underlying the formation of ERPO. Other ER derived degradation subcompartments include a quality control compartment for misfolded glycoproteins, termed ERQC  and a compartment termed ERAC (ER associated compartment) that is formed in yeast expressing a multispanning membrane protein (Ste6p) with mutation in cytoplasmatic domains . ERAC comprises a network of tubulo-vesicular structures that occasionally are continuous with the ER profiles, and was found to prevent proteins targeted for ERAD from entering the secretory pathway . These properties are compatible with features of VAPB inclusions. A hallmark of our VAPB inclusions is their position in the center of normal appearing RER that in motor neurons usually is organized in Nissl Bodies  (Figure 2). This position indicates that mutant VAPB is sorted in a direction opposite to the secretory pathway. Further work is needed to determine whether mutant VAPB inclusions represent an ERAC-like compartment, and whether there are additional ER substrates with misfolded cytosolic domains that accumulate in the same structures. Our finding that mutant VAPB with a different ER transmembrane domain accumulate in the same structures (Figure 7J, K) favors the notion that the mutant VAPB inclusions represent a protective ‘waste basket’ for multiple ERAD-C substrates.
The molecular mechanisms underlying the formation of mutant VAPB inclusions remain to be further determined. Previously, we have shown that their formation does not require microtubule-dependent transport, which is instrumental for the formation of aggresomes and several other quality control compartments [4, 6]. Here, we show that shRNA-mediated knock-down of the ER membrane bound E3 ligase TEB4 (MARCH-VI) severely reduces the size and number of mutant VAPB inclusions. TEB4 and its yeast ortholog Doa10, mediate ubiquitination of multiple ERAD-C substrates [53, 54], and accordingly TEB4 may mediate ubiquitination of mutant VAPB to target it for the ERAD machinery [54, 62–64]. These data suggest that ubiquitination or another activity of TEB4 is an early step in the formation of VAPB inclusions. We also found that knock-down of the ER membrane chaperone BAP31 increases the size of VAPB inclusions. BAP31 has been implicated in ERAD [24, 51], accumulates in the mutant VAPB inclusions, and may be involved in the interaction of ubiquitinated mutant VAPB with VCP. In this scenario the absence of BAP31 would prevent efficient extraction of mutant VAPB from the ER membrane by VCP. Some proteins that interact with the transmembrane domain of mutant VAPB, e.g. wild-type VAPs  and YIF1A , are recruited to the VAPB inclusions, raising the question what happens with these proteins during ERAD of mutant VAPB. Another question is how the formation of ER quality control compartments such as VAPB inclusions, are connected to unfolded protein response pathways that may be activated in conditions of proteotoxic ER stress and overload of ERAD, and that have been implicated in ALS pathogenesis .
Wild-type VAPB overexpression causes stacked ER
Recent work of Borgese and co-workers [5, 52] suggests that VAPB-P56S inclusions in HeLa cells predominantly consist of a special form of stacked ER, made of two or three tightly apposed ER cisternae separated by an electron-dense layer [5, 52]. We did not observe this or any other form of stacked ER in our mutant VAPB expressing lines. VAPB inclusions in HeLa cells are also BAP31  and VCP/p97-positive (data not shown), and mutant VAPB was shown to be rapidly ubiquitinated, and degraded in a proteasome and VCP/p97 dependent way , suggesting that as in neurons mutant VAPB inclusions may represent an ERAD-associated compartment. Renewed ultrastructural analysis of our HeLa cells showed that in cells with relatively few and small inclusions they resembled the inclusions observed in mutant VAPB mice (Additional file 1: Figure S6A, B), while in cells with more and larger inclusions they showed more complex morphologies (Additional file 1: Figure S6C-E). Interestingly, we noted patches of apposed ER cisternae separated by a thin layer of electron dense cytosol, resembling the apposed ER cisternae reported by Borgese and coworkers. However, so far in our HeLa cells we have not identified the relatively large domains of bi- or trilaminar ER documented by Borgese et al. [5, 52]. Hence, the precise relationship between our VAPB inclusions and the remodeled stacked ER of Borgese et al. remains to be determined.
Stacked ER occurred in motor neurons of our wild-type hVAPB transgenic mice, and is a well-documented phenomenon in cells that coexpress wild-type VAPB and FFAT-motif proteins, presumably resulting from heterotypic interaction between VAPB and FFAT-motif proteins [41, 42]. Hence, stacked ER in motor neurons of wild-type VAPB overexpressing mice may result from excessive VAPB interacting with endogenous FFAT-motif protein. Remarkably, in one line of wild-type VAPB overexpressing mice we observed a variant of stacked ER where the cytosolic space linking the cisterns was considerably more electron dense. These data indicate that stacked ER in some conditions is irreversible, which contrasts with the notion that stacked ER is a relatively harmless and reversible phenomenon [39, 40].
How does mutant VAPB cause motor neuron disease?
Consistent with previous studies [17, 18] our data show that neuron-specific mutant VAPB transgenic mice generally do not develop motor symptoms and signs of motor neuron degeneration. This contrasts with the pathological phenotypes observed in drosophila expressing mutant VAPB (DVAP-P58S or DVAP-T48I) that develop loss of function-like phenotypes, and are suggestive of a dominant-negative mode of action of mutant VAPB [6, 7, 19, 49, 67]. The absence of a phenotype in mice may be explained by efficient degradation and the accumulation of mutant VAPB in a protective compartment that prevents mutant VAPB from accumulating at sites where it could engage in aberrant interactions such as the ER Golgi intermediate compartment (ERGIC), or the axon. The same mechanisms may explain the late and variable onset of disease in man [8, 68, 69]. Interestingly, two (out of 46) mice of our aging cohort developed a late onset motor axonopathy that is reminiscent of mutant VAPB induced disease in man. Although their number is too low to draw conclusions, a striking feature of these mice with motor axon pathology was the absence of VAPB inclusions, indicative of a negative correlation between the presence of inclusions and the development of pathology. However, in view of our finding that inclusions gradually disappear in axotomized motor neurons, an alternative explanation is that the inclusions in the mice with motor axon pathology have disappeared secondarily to the axonal pathology. Further work is needed to determine the role of protective pathways like ERAD and the formation of inclusions in preventing disease onset in mutant VAPB expressing mice, as well as the factors that cause the disappearance of VAPB inclusions after axotomy.
A recent study with Vapb −/− mice has indicated that VAPB deficiency leads to mild, late onset defects in motor performance, but does not cause neuromuscular junction abnormalities and muscle denervation . These data suggest that loss of VAPB function by itself is not sufficient to trigger an ALS-like disorder perhaps because of compensatory actions by VAPA  and point to alternative or additional mechanisms for mutant VAPB toxicity, such as a gained toxic activity, or a dominant-negative effect. It would be interesting to cross VAPB-P56S transgenic mice with Vapb −/− mice to examine the presence of synergistic deleterious interactions between the absence of VAPB and the presence of mutant VAPB.
In conclusion, the central finding of the present study is that inclusions formed by ALS8-mutant VAPB in motor neurons in transgenic mice represent a protective ER compartment that isolates misfolded and aggregated VAPB from the rest of the ER. The data suggest that motor neurons are capable of coping with mutant VAPB levels that exceed the capacity of the ERAD systems. Whether similar protective ER derived compartments occur in physiological and pathological conditions in human central nervous system could be analyzed by immunohistological approaches with antibodies against BAP31 and VCP/p97.
This research was supported by Prinses Beatrix Spierfonds grant (DJ, CH), Hersenstichting Nederland (DJ). The ALS Association (CH, DJ), Netherlands Organization for Scientific Research (NWO-ALW-VICI, CH; NWO-ALW VENI, MH), Cyttron II (FES0908, KV, JAP). The authors would like to thank Dr N. Ridgway (Dalhousie University, Canada) and Dr. V. Olkkonen (Institute for Molecular Medicine Finland) for anti-ORP antibodies; Dr. M. Tagaya (Tokyo University of Pharmacy and Life Sciences) for the BAP31-mRFP construct and BAP31 antibody; and Dr. D. Ito (School of Medicine, Keio University, Tokyo, Japan) for seipin constructs; and Drs. F. Navone, M. Francolini; and N. Borgese for showing preliminary data and helpful discussions (CNR Inst Neuroscience, Milano; Italy).
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