VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that...  Read more

VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that correlates with an increase in post-synaptic glutamate receptor clustering. It has also been shown that hVAPB, the causative gene of ALS8, rescues the lethality and the neuromuscular junction phenotype associated with loss of DVAP, clearly indicating that the fly protein and human VAP perform homologous functions (3).

Recently, reports from two independent labs (Tsuda et al. and Ratnaparkhi et al.) have provided new and exciting insight on the normal function of VAP proteins and their possible role in the pathogenesis of VAP-induced ALS. Comments on these papers can be summarized as follows.

The paper by Tsuda et al. reports that VAP proteins are cleaved, and an N-terminal fragment of a size compatible with the size of the MSP domain is secreted and binds to the Eph receptors. The pathogenic allele induces the accumulation of the mutant and the wild-type (wt) protein into the ER and a failure to secrete the cleaved MSP domain. Non cell-autonomous effects of the mutant and wt proteins have been reported both at the level of the Drosophila nervous system and the nematode reproductive system. The ability of dVAP to be cleaved and secreted has been shown with an elegant experiment in which the expression of dVAP has been driven in a subset of cells in the wing imaginal discs. A diffusion of dVAP MSP beyond the protein expressing cells was observed. However, there is no direct evidence that this process of cleavage and secretion of VAP proteins is occurring in neurons, in muscles, or in any other tissue that would be more relevant to the human disease.

The ability of the pathogenic allele to induce the formation of aggregates has been previously reported in cell culture (1,4,5) and Drosophila model systems (3). Tsuda and colleagues report that expression of the mutant protein in a null background induces the formation of detergent-insoluble aggregates. Despite the mutant allele being inherited in a dominant manner in humans, these data lead to the important conclusion that the wild-type protein is not necessary for the formation of aggregates. However, an intriguing question arises: how can the presence of these aggregates be reconciled with the ability of the mutant protein to rescue the phenotypes associated with null mutations in dVAP as shown by three independent studies (3, Ratnaparkhi et al., Tsuda et al.). Are these aggregates different from the ones observed when the mutant protein is expressed in the presence of the wt protein?

Other outstanding questions will need to be addressed: which is the protease or proteases responsible for the cleavage? Is the secretion of the MSP domain of VAP proteins occurring through an unconventional mechanism as already proposed for the MSP proteins in C. elegans? Which is the subcellular compartment in which the cleavage occurs?

The paper by Ratnaparkhi et al. focuses on another important aspect, which is the determination of the disease mechanism. In humans, the pathogenic mutation is inherited in a dominant manner. Dominant mutations are due to a gain of function (hypermorphs and neomorphs), dominant-negative interactions (antimorphs) or haplo-insufficiency. Understanding the patho-mechanism of the disease is important as it can indicate new possible strategies for therapeutic interventions. Several lines of evidence support a possible dominant-negative effect of the pathogenic allele. The formation of aggregates, the depletion of the wild-type protein from its normal localization (3,4,5), and the sequestration of the wt protein in the aggregates clearly suggest a dominant-negative effect (4). Moreover, the fact that the pathogenic allele acts as a dominant-negative can be proven if the overexpression of the mutant protein in the presence of the wt protein leads to a phenotype similar to the loss-of-function mutation. Indeed, it has been reported that transgenic expression of the mutant protein induces a reduction in number of boutons (3), a disruption of the presynaptic cytoskeleton (Ratnaparkhi et al.) and a reduction in miniature excitatory post-synaptic potentials (Tsuda et al.). Ratnaparkhi et al. attempt to further support this statement by performing a systematic analysis of mutant phenotypes in different functional contexts. They compared the effect of overexpressing the wt protein with the overexpression of the mutant protein in transgenic lines expressing comparable amounts of transgenes. The expression levels of the proteins were estimated only for the full-length VAP. Although the mutant allele impairs the secretion of the MSP domain, the cleaved product is still produced as shown in several Western blots reported by Tsuda et al. The same Western blots suggest that the levels of the full-length protein and the cleaved MSP domain are not stoichiometrically similar; therefore, restricting the analysis to the expression levels of the full-length protein may be misleading. A cleaved, non-secreted MSP domain could still be responsible for the intracellular, cell-autonomous effects of the protein.

Although there are several lines of evidence supporting a possible dominant-negative effect, there is other evidence suggesting different mechanisms for the disease. Mutant VAP proteins still retain some functional properties of the wt protein such as the ability to self-oligomerize (3,4) and the ability to rescue, at least in part, the mutant phenotype due to the loss of the endogenous protein. The mutant allele has also acquired new functional properties that are not shared by the normal version of the protein such as the propensity to form aggregates and the “floating active zones” phenotype reported by Ratnaparkhi et al. In one report it has also been shown that the mutant protein has an increased ability of inhibiting the activity of ATF6, a transcription factor involved in UPR (6).

We propose that the mutant allele may cause the disease by a combination of mechanisms that include dominant-negative interactions and toxic effects due to gain of new functions. Although a lot still remains to be done, studies published over the last six months have convincingly shown that the variety of genetic tools available in Drosophila can now be exploited to foster our understanding of the patho-mechanisms responsible for motor neuron diseases in humans.

References:
1. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

2. Pennetta G, Hiesinger PR, Fabian-Fine R, Meinertzhagen IA, Bellen HJ. Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron. 2002 Jul 18;35(2):291-306. Abstract

3. Chai A, Withers J, Koh YH, Parry K, Bao H, Zhang B, Budnik V, Pennetta G. hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet. 2008 Jan 15;17(2):266-80. Abstract

4. Kanekura K, Nishimoto I, Aiso S, Matsuoka M. Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J Biol Chem. 2006 Oct 6;281(40):30223-33. Abstract

5. Teuling E, Ahmed S, Haasdijk E, Demmers J, Steinmetz MO, Akhmanova A, Jaarsma D, Hoogenraad CC. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J Neurosci. 2007 Sep 5;27(36):9801-15. Abstract

6. Gkogkas C, Middleton S, Kremer AM, Wardrope C, Hannah M, Gillingwater TH, Skehel P. VAPB interacts with and modulates the activity of ATF6. Hum Mol Genet. 2008 Jun 1;17(11):1517-26. Abstract

View all comments by Giuseppa Pennetta

The paper by Tsuda et al. in the latest issue of Cell makes the very interesting suggestion that a Drosophila VAP protein is cleaved, released into the extracellular space, and activates Eph receptors.

The evidence that the protein is cleaved, or proteolyzed, is based on the immunoblot detection of truncated forms of VAP in wild-type animals and transgenic flies expressing epitope tagged forms of dVAP (dVAP33A). This is consistent with what has previously been shown for the two rodent VAP proteins VAPA and VAPB (1,2). The authors then suggest that a similar truncated form of VAP can be found in human serum. The evidence for this is less compelling. The species detected in human serum is clearly larger than the truncated fragment detected in white blood cells. In addition, this anti-sera was raised to full-length VAPB, and there is no evidence to indicate it is recognizing the MSP domain. It is notable that a number of polyclonal anti-sera raised against full-length recombinant VAPA and VAPB are specific for each protein. Given the near identical structure of the MSP...  Read more

The paper by Tsuda et al. in the latest issue of Cell makes the very interesting suggestion that a Drosophila VAP protein is cleaved, released into the extracellular space, and activates Eph receptors.

The evidence that the protein is cleaved, or proteolyzed, is based on the immunoblot detection of truncated forms of VAP in wild-type animals and transgenic flies expressing epitope tagged forms of dVAP (dVAP33A). This is consistent with what has previously been shown for the two rodent VAP proteins VAPA and VAPB (1,2). The authors then suggest that a similar truncated form of VAP can be found in human serum. The evidence for this is less compelling. The species detected in human serum is clearly larger than the truncated fragment detected in white blood cells. In addition, this anti-sera was raised to full-length VAPB, and there is no evidence to indicate it is recognizing the MSP domain. It is notable that a number of polyclonal anti-sera raised against full-length recombinant VAPA and VAPB are specific for each protein. Given the near identical structure of the MSP domains, this lack of cross-reactivity may indicate that this domain is poorly immunogenic.

The presence of dVAP immunoreactivity on the surface of the cell is particularly interesting. Most previous studies have concentrated on VAP proteins present on intracellular membranes such as the ER and Golgi. However, Lapierre et al. (3) have reported that in liver the majority of VAPA was found associated with the plasma membrane, where it interacted with occludin. An important unresolved issue is what proportion of dVAP is actually secreted on the surface, cleaved, or associated with cellular membranes. How the protein gets to the surface of the plasma membrane and out of the cell is yet to be determined. Perhaps it has something to do with the ability of VAP proteins to generate multi-lamella membranes when expressed in certain contexts (4)?

Interest in VAP proteins was stimulated greatly by the discovery of a familial form of motor neuron disease associated with a missense mutation in VAPB (5). Tsuda et al. demonstrate that the analogous mutation in dVAP, P58S, blocks the delivery of the protein to the surface. Moreover, the mutant protein is shown to associate with the wild-type protein as ubiquitinated aggregates within cells. This is consistent with the mutant protein aggregates described originally and with recent work that reported similar ubiquitinated complexes containing both mutant and wild-type proteins in vertebrate cells (5-7). Similarly, the induction of the unfolded protein response seen in Drosophila overexpressing wild-type or mutant dVAP is consistent with previous findings in vertebrates. However, the exact relationship of VAP proteins with ER stress regulation may be more complicated, as they appear to both increase and reduce different pathways of this regulatory system (1,6,7).

Whether the protein is cleaved before or after it is secreted is not directly examined. However, since the P58S mutation is retained within cells yet is still cleaved, it seems reasonable to suggest that the cleavage occurs before the MSP domain would be released. How the MSP domain remains associated with the cell membrane in such circumstances is not clear.

The potential link between Eph receptors and VAP proteins is very exciting. The structure of the MSP domain of VAP proteins and the C. elegans MSP are very similar (8), and the extracellular signaling properties of the Drosophila and human MSP domains expressed in isolation are demonstrated by the ability to mimic C. elegans MSP induced oocyte maturation and sheath contraction. It will be very interesting to see if the C. elegans VAP protein also contributes to the MSP-mediated oocyte maturation process.

In summary, this report builds upon previous work on the MSPs of C. elegans and on vertebrate and Drosophila VAP proteins, and implicates the Eph/Ephrin pathway in the neurodegenerative processes of motor neuron disease. It also suggests that VAP proteins may be trafficked in cells by previously unappreciated processes. If VAP proteins are shown to have similar properties in vertebrates, then this work may have highlighted a new pathway for therapeutics against motor neuron disease.

References:
1. Gkogkas C, Middleton S, Kremer AM, Wardrope C, Hannah M, Gillingwater TH, Skehel P. VAPB interacts with and modulates the activity of ATF6. Hum Mol Genet. 2008 Jun 1;17(11):1517-26. Abstract

2. Skehel PA, Fabian-Fine R, Kandel ER. Mouse VAP33 is associated with the endoplasmic reticulum and microtubules. Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1101-6. Abstract

3. Lapierre LA, Tuma PL, Navarre J, Goldenring JR, Anderson JM. VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction. J Cell Sci. 1999 Nov;112 ( Pt 21):3723-32. Abstract

4. Amarilio R, Ramachandran S, Sabanay H, Lev S. Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction. J Biol Chem. 2005 Feb 18;280(7):5934-44. Abstract

5. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

6. Kanekura K, Nishimoto I, Aiso S, Matsuoka M. Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J Biol Chem. 2006 Oct 6;281(40):30223-33. Abstract

7. Teuling E, Ahmed S, Haasdijk E, Demmers J, Steinmetz MO, Akhmanova A, Jaarsma D, Hoogenraad CC. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J Neurosci. 2007 Sep 5;27(36):9801-15. Abstract

8. Kaiser SE, Brickner JH, Reilein AR, Fenn TD, Walter P, Brunger AT. Structural basis of FFAT motif-mediated ER targeting. Structure. 2005 Jul;13(7):1035-45. Abstract

View all comments by Paul Skehel

Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important...  Read more

Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important to study a mutation which is only responsible for a small percentage of ALS cases?

One reason is due to the fact that from a clinical point of view, familial and sporadic ALS cases are virtually identical. As such, it is not unreasonable to postulate that although ALS may be caused by different genetic factors, they all may lead to common sets of pathways that eventually result in the ALS phenotype. Thus, a high level of importance should be placed on understanding the common features of all known ALS genes since they may shed light on these pathways. Therefore, even though VAPB mutations are indeed rare, characterizing their effects may provide insight on how cases of ALS develop overall.

In both of the papers presented (6,7), the authors have each developed a Drosophila model of ALS which expresses mutant VAPB. The use of these models will undoubtedly be beneficial in future experiments to further decipher the ALS phenotype. Of great significance, though, is that each study observes in vivo that mutant VAPB is capable of inducing intracellular aggregates. This work reinforces previously published observation that in vitro expression of mutant human P56S protein results in cellular aggregates (4,5). The fact that the aggregation phenotype of this mutation is conserved down to Drosophila is quite interesting. Aggregates are commonly observed within ALS cases, as well as other neurodegenerative diseases, although whether these aggregates are pathogenic is still up for debate. The formation of intracellular aggregates has also been observed via expression of mutant SOD1 and mutant TDP-43 (3). Taken together, the observation that three different familial ALS genes all are capable of inducing intracellular aggregates reinforces the notion that understanding the activation of pathways by protein misfolding is key to understanding the pathogenic nature of ALS.

References:
1. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Abstract

2. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande Velde C, Bouchard JP, Lacomblez L, Pochigaeva K, Salachas F, Pradat PF, Camu W, Meininger V, Dupre N, Rouleau GA. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May;40(5):572-4. Abstract

3. Winton MJ, Van Deerlin VM, Kwong LK, Yuan W, Wood EM, Yu CE, Schellenberg GD, Rademakers R, Caselli R, Karydas A, Trojanowski JQ, Miller BL, Lee VM. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett. 2008 Jun 25;582(15):2252-6. Abstract

4. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

5. Landers JE, Leclerc AL, Shi L, Virkud A, Cho T, Maxwell MM, Henry AF, Polak M, Glass JD, Kwiatkowski TJ, Al-Chalabi A, Shaw CE, Leigh PN, Rodriguez-Leyza I, McKenna-Yasek D, Sapp PC, Brown RH Jr. New VAPB deletion variant and exclusion of VAPB mutations in familial ALS. Neurology. 2008 Apr 1;70(14):1179-85. Abstract

6. Tsuda H, Han SM, Yang Y, Tong C, Lin YQ, Mohan K, Haueter C, Zoghbi A, Harati Y, Kwan J, Miller MA, Bellen HJ. The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell. 2008 Jun 13;133(6):963-77. Abstract

7. Ratnaparkhi A, Lawless GM, Schweizer FE, Golshani P, Jackson GR. A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism. PLoS ONE. 2008 Jun 4;3(6):e2334. Abstract

View all comments by John Landers