InteractiveFly: GeneBrief

VAMP-associated protein 33kDa: Biological Overview | References

BIOLOGICAL OVERVIEW

VAP proteins (human VAPB/ALS8, Drosophila VAP33, and C. elegans VPR-1) are homologous proteins with an amino-terminal major sperm protein (MSP) domain and a transmembrane domain. The MSP domain is named for its similarity to the C. elegans MSP protein, a sperm-derived hormone that binds to the Eph receptor (see Drosophila Eph) and induces oocyte maturation. A point mutation (P56S) in the MSP domain of human VAPB is associated with Amyotrophic lateral sclerosis (see Drosophila as a Model for Human Diseases: Amyotrophic lateral sclerosis), but the mechanisms underlying the pathogenesis are poorly understood. This study shows that the MSP domains of VAP proteins are cleaved and secreted ligands for Eph receptors. The P58S mutation in VAP33 leads to a failure to secrete the MSP domain as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response. It is proposed that VAP MSP domains are secreted and act as diffusible hormones for Eph receptors. This work provides insight into mechanisms that may impact the pathogenesis of ALS (Tsuda, 2008).

The mechanisms that underlie ALS are poorly understood. ALS is associated with the dysfunction or death of motor neurons in the motor cortex, brain stem, and spinal cord. About 10%-15% of all ALS cases are familial, whereas 85%-90% are sporadic. The most common form of familial ALS is caused by mutations in superoxide dismutase 1 (SOD1). Recently, Nishimura (2004) identified another gene, ALS8, that causes familial ALS. This gene encodes the VAMP (synaptobrevin)-associated protein B (VAPB). Lesions in SOD1 and ALS8 have been shown to cause a wide variety of symptoms that typically include motor neuron death, but vary widely in the age of onset, the speed of progression, and the motor neuron populations that are affected. For example, a single amino acid change in ALS8 (P56S) causes typical ALS, atypical slowly progressive ALS, and spinal muscular atrophy (SMA) with an age of onset between 25 and 52 years and a speed of progression between 2 and 30 years (Nishimura, 2004). The cause of this variation may be due to genetic modifiers, partial redundancy, or environment (Tsuda, 2008).

VAPB is closely related to VAPA, which has been shown to associate with the cytoplasmic face of the endoplasmic reticulum (ER) and the Golgi apparatus (Kaiser, 2005; Skehel, 2000; Soussan, 1999). Human VAPB (hereafter named hVAP) protein is about 30 kDa and has homologs in C. elegans (VPR-1), Drosophila (VAP33-A, hereafter named dVAP) (Pennetta, 2002), and numerous other species, including yeast (Scs2p) (Kagiwada, 2003). VAPs consist of an amino (N)-terminal domain of about 125 residues called the major sperm protein (MSP) domain, which is conserved among all VAP family members (Nishimura, 1999; Weir, 1998). The central region is predicted to form a coiled-coil motif. The hydrophobic carboxy (C)-terminus acts as a membrane anchor. The MSP domain is named for its similarity to nematode MSPs, the most abundant proteins in nematode sperm (Bottino, 2002). MSP and VAP MSP domains fold into evolutionarily conserved immunoglobulin-type seven-stranded β sandwiches (Baker, 2002; Kaiser, 2005), suggesting a common function (Tsuda, 2008).

The main difference between VAPs and MSP is their proposed functions. C. elegans MSPs do not contain a coiled-coil motif or a transmembrane domain (Ward, 1988). MSPs have an intracellular cytoskeletal function, which depends on their ability to polymerize in the absence of actin or myosin (Bottino, 2002) and an extracellular signaling function during fertilization (Miller, 2001). MSP is secreted from the sperm cytosol into the reproductive tract by an unconventional process (Kosinski, 2005). Extracellular MSP directly binds to the VAB-1 Eph receptor and other yet-to-be-identified receptors on oocyte and ovarian sheath cell surfaces (Corrigan, 2005: Govindan, 2006; Miller, 2003). MSP induces oocyte maturation, which prepares oocytes for fertilization and embryogenesis, and sheath contraction (Miller, 2001; Tsuda, 2008 and references therein).

The Eph receptors are an evolutionarily conserved class of receptor tyrosine kinases that bind to membrane-attached ligands called Ephrins. Ephrins act in parallel to gap junctions to inhibit oocyte maturation, and MSP antagonizes this inhibitory circuit (Govindan, 2006; Miller, 2003; Whitten, 2007). MSP induces activation of the MAP kinase and Ca²⁺/calmodulin-dependent protein kinase II cascades (Corrigan, 2005; Miller, 2001) as well as reorganization of the oocyte microtubule cytoskeleton (Harris, 2006; Tsuda, 2008 and references therein).

The biological function of VAPs is not well understood. Yeast Scs2p is involved in phosphatidylinositol-4-phosphate synthesis and ceramide transport (Brickner, 2004; Kagiwada, 2003). VAPs have been reported to associate with the ER (Amarilio, 2005; Kaiser, 2005; Soussan, 1999). Overexpression of hVAP in human cells affects the structural integrity of the ER (Teuling, 2007) through interaction with Nir (N-terminal domain-interacting receptor) proteins (Amarilio, 2005). VAPs also interact with oxysterol-binding protein (OSBP) and ceramide transfer protein. These interactions are each mediated through FFAT (two phenylalanines in an acidic tract) domains (Amarilio, 2005; Kaiser, 2005; Loewen, 2005). Taken together, the results suggest that VAPs might play a role in fatty acid metabolism (Perry, 2006; Wang, 2005; Tsuda, 2008 and references therein).

To further define the role of VAPs, Drosophila dVAP (Pennetta, 2002) has been characterized. dVAP modulates the number and size of neuromuscular junction (NMJ) boutons. Loss of dVAP disrupts the presynaptic microtubule architecture (Pennetta, 2002) and causes an increase in miniature excitatory junctional potential (mEJP) size as well as an increase in postsynaptic glutamate receptor clustering (Chai, 2008; Tsuda, 2008).

This study presents evidence that VAP MSP domains are secreted ligands for Eph receptors. It is proposed that secreted MSP domains function as trophic factors by binding to Eph receptors and other cell-surface receptors. The P56S mutation that causes ALS8 (P58S in dVAP) induces insoluble aggregates that are ubiquitinated in flies. The mutation also leads to an accumulation of mutant and wild-type protein in the ER, an unfolded protein response (UPR), and a failure to secrete the MSP domain. Collectively, these results suggest that P56S affects a cell-autonomous pathway involving the ER and UPR as well as a cell nonautonomous pathway involving Eph receptor signaling (Tsuda, 2008).

ALS is a disease caused by death of anterior horn motor neurons in the spinal cord and neurons in motor cortex, after decades of apparently normal development and function. Familial and sporadic ALS cases as well as mouse models induced by overexpressing mutant SOD1 indicate that all forms lead to intracellular cytoplasmic protein inclusions containing ubiquitinated proteins. In flies expressing P58S dVAP, cytoplasmic inclusions and other key characteristics of ALS were found. (1) P58S dVAP protein induces ubiqutinated inclusions. (2) The protein inclusions are associated with the ER and appear to be electron-dense ER expansions. (3) Several key ER proteins colocalize with these inclusions. Finally, mutant dVAP induces a unfolded protein response (UPR). These data show at least three important parallels with ALS and SOD1 mouse models: cytoplasmic inclusions, ubiquitination, and the UPR. The UPR-induced stress caused by P58S dVAP could eventually result in cellular damage or neuronal death (Chai, 2008; Tsuda, 2008).

Another feature associated with ALS is that the disease may have a cell-non-autonomous component. VAP MSP domains can be secreted, although not all cell types appear capable of secretion in flies. The VAP proteins, including the yeast homolog SCS2, have been proposed to be type II-membrane proteins (Kagiwada, 1998). Since the proteins lack an N-terminal signal sequence, similar to MSP, secretion is likely to occur by an unconventional mechanism as observed for the C.elegans MSP proteins (Kosinski, 2005). In addition, the hVAP MSP domain is present in blood serum). The MSP in serum may be able to bind to Eph receptors present on endothelial cells, which regulate angiogenesis (Kuijper, 2007). Indeed, SOD1 mutants display defects in the tight junctions between endothelial cells, and endothelial damage occurs prior to motor neuron degeneration. Interestingly, Teuling (2007) recently reported that VAPB is significantly decreased in the spinal cord of SOD1 mutants and human patients with sporadic ALS. It is therefore possible that reduced signaling by the hVAP MSP domain is a mechanism responsible for some nonautonomous features associated with ALS pathogenesis (Tsuda, 2008).

This study shows that secreted MSP domains bind to Eph receptors on the surfaces of cells. Eph receptors also bind to ligands called Ephrins. MSP domains function in vivo to antagonize Ephrin signaling during oocyte maturation and, possibly, amphid neuron migration. Competition assays are consistent with MSP domains competing with Ephrin for Eph receptor binding. In other processes, including worm-DTC cell migration, ovarian sheath contraction, and fly MB formation, MSP domains seem to be required for Eph receptor signaling (Miller, 2003). Hence, the relationship between MSP and Ephrin ligands to Eph receptor signaling may depend on the developmental context, as previously observed for Ephrins and Eph receptors in mammals (Himanen, 2007). Multiple Ephrins and Eph receptors including EphA4 and A7 are expressed throughout the adult nervous system and in skeletal muscle of vertebrate species. Eph receptors regulate the survival of cultured spinal cord motor neurons and influence proliferation and apoptosis in the adult mammalian CNS. VAP MSP may play a role in motor neuron survival or muscle function through interactions with Eph receptors (Tsuda, 2008).

Glutamate excitotoxicity is likely to play a role in the pathogenesis of ALS (Bruijn, 2004). Three lines of evidence suggest that VAP MSP domains might regulate glutamate receptor signaling. (1) Eph receptors directly associate with NMDA-subtype glutamate receptors and regulate clustering in cultured neurons (Dalva, 2000). (2) Loss of dVAP function or overexpression of P58S in flies is associated with increased glutamate receptor clustering and increased amplitudes of mEJPs at the NMJs (Chai, 2008). (3) MSP and the VAB-1 Eph receptor regulate NMDA receptor function during worm oocyte maturation (Corrigan, 2005; Tsuda, 2008 and references therein).

The following model is proposed for the pathogenesis of ALS8. The P56S hVAP protein accumulates in the ER, while the wild-type protein is functional. In time, the aggregates become more prominent, P56S hVAP becomes ubiquitinated, and functional wild-type proteins become trapped in the inclusions. These protein inclusions initiate a UPR that eventually affects cell viability and lead to a decrease in MSP domain secretion. Impaired secretion decreases signaling by Eph receptors and other receptors. The mutant protein therefore causes two different defects: a cell-autonomous defect in the ER that creates a UPR and a cell non-autonomous defect resulting from reduced secretion of VAP MSP, which may function as an autocrine or paracrine signal. Both defects may synergize to produce the key features of ALS pathology. This model provides testable hypotheses and raises questions to be addressed in the future (Tsuda, 2008).

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

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of the fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans (Chai, 2008).

hVAPB has been shown to be the causative gene of late-onset autosomal dominant forms of motor neuron disorders, including typical and atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation predicts a substitution of a Serine for a conserved Proline (P56). One of the hallmarks associated with loss-of-function and neuronal overexpression of DVAP-33A is decreased and increased bouton formation at the NMJ, respectively. Despite this structural alteration, synaptic transmission is maintained within a wt range. At the mechanistic level, muscles respond to a decreased number of boutons and quantal content by upregulating quantal size; conversely muscles compensate an increase in number of boutons and quantal content by downregulating quantal size. Compensatory changes in quantal size during synaptic homeostasis are thought to be determined, largely, by the properties of transmitter receptors. At the Drosophila NMJ, there are two classes of glutamate receptors: one set containing the subunit IIA and another one containing the subunit IIB. In DVAP-33A loss-of-function mutations, the increase in quantal size is associated with an increase in the number and average cluster volume of subunit IIA. Conversely, the decrease in quantal size in the oversprouting mutants is accompanied by a decrease in the level of post-synaptic receptor subunit IIA and a reduction in the average cluster volume for several subunits. In agreement with these data, the IIA subunit receptors have been shown to affect quantal size and receptor channel open time. Similar to the oversprouting mutants, in synapses lacking the receptor subunit IIA, a homeostatic increase in neurotransmitter release compensates for the reduction in quantal size and the evoked response is maintained within normal values. These data indicate that expression levels of VAP proteins play a crucial role in synaptic homeostasis by coordinating structural remodeling and post-synaptic sensitivity to neurotransmitter to ensure synaptic efficacy (Chai, 2008).

Interestingly, expression of hVAPB in neurons rescues lethality, morphological and electrophysiological phenotypes associated with DVAP-33A loss-of-function mutations. Moreover, neuronal expression of hVAPB in a wt background induces phenotypes similar to the overexpression of DVAP-33A. These data clearly indicate that DVAP-33A and hVAPB perform homologous functions at the synapse and as a consequence, information gained by studying DVAP-33A is expected to be relevant for hVAPB function as well. Surprisingly, neuronal expression of mutant VAP proteins also rescues all phenotypes associated with mutations in DVAP-33A. Two alternative scenarios could be proposed to explain these data: the mutation is irrelevant for the ALS8 pathogenesis or the mutant allele has a pathogenic effect while retaining certain functional properties of the wt protein. The second hypothesis is favored for the following reasons. (1) The P56S mutation in hVAPB has been reported to be causative for an inherited form of MNDs in humans. This mutation affects nine related families totaling 1500 individuals of which 200 suffer from motor neuron disorders. (2) A genetic model for MNDs was generated where the expression of the aberrant VAP recapitulates major hallmarks of the human disease, clearly indicating that the mutation has a pathogenic effect. (3) The data suggest that both the Drosophila and the human mutant proteins retain some functional wt properties such as the ability to self-oligomerize. However, neuronal expression of the pathogenic protein induces aggregate formation and depletes the wt protein from its normal localization. These effects are not observed when the wt protein is overexpressed, suggesting that the mutant protein has acquired a new, potentially toxic property (Chai, 2008).

Indeed, one of the most common features of MNDs and nearly all neurodegenerative diseases is the accumulation of aggregates that are intensively immuno-reactive to disease-related proteins. Each disease, however, differs with respect to the anatomical location and morphology of the aggregates. The major component of the aggregates is usually the protein encoded by the gene mutated in the familial forms, which is also unique to each disease. Despite this diversity, a bulk of circumstantial evidence support the hypothesis that aggregates are typical hallmarks of neurodegenerative diseases and have a toxic effect on neurons. While no autopsy material is available for familial cases with the P56S mutation, SOD1-positive inclusions have been reported in human sporadic and familial ALS cases as well as in SOD1 mouse models. This study found the presence of aggregates that are intensively immuno-reactive for DVAP-33A both in neuronal cell bodies and in nerve fibers of the MND model. Interestingly, hVAPB carrying the pathogenic mutation has also been shown to undergo intracellular aggregation when expressed in a cell culture system. However, similarities between human disease and the fly model are not limited to aggregate formation as flies expressing transgenic VAP proteins carrying the ALS8 mutation, exhibit other hallmarks of the human disease such as neuronal cell death, muscle wasting and defective locomotion behavior (Chai, 2008).

Although it remains to be established whether the VAP protein in the aggregates represents the mutant protein, the endogenous protein or a mixture of both, a regional decrease in the level of the endogenous protein is clearly observed. The DVAP-33A protein that is normally associated with the plasma membrane in neuronal cell bodies and at the neuromuscular synapses is nearly undetectable in DVAPP58S transgenic animals. As a consequence of the decrease in synaptic levels of the endogenous protein, a decrease in the number of boutons is observed. It has been previously shown that DVAP-33A regulates bouton formation at the synapse in a dosage-dependent manner. Despite these structural alterations a homeostatic mechanism is established to maintain synaptic efficacy within functional boundaries. It is speculated that the depletion of the endogenous protein from its normal localization and the formation of aggregates would affect the homeostatic mechanism linking structural remodeling and synaptic efficacy controlled by DVAP-33A. Although not directly tested in this model, experiments in cell culture show that overexpression of mutant hVAPB induces formation of aggregates in which the endogenous wt protein is recruited. This would suggest that the pathogenic allele functions as a dominant negative. However, the depletion of the endogenous protein from its normal localization cannot be the principal mechanism of the disease as mutants lacking DVAP-33A do not develop MND. It is therefore possible that the pathogenic allele has acquired an abnormal, new toxic activity. Similar to what has been proposed for other neurodegenerative diseases, the formation of aggregates may directly interfere with critical cellular processes and/or compromise the ability of the system to keep up with the degradation of aggregated proteins (Chai, 2008).

Taken together these data offer experimental support to the hypothesis that VAP proteins play a conserved role in synaptic homeostasis and emphasize the relevance of this fly model in fostering an understanding of the molecular mechanisms underlying VAP-induced motor neuron degeneration in humans (Chai, 2008).

VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. This study found that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. Loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. It is proposed that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation (Mao, 2019).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive motor neuron degeneration and muscle weakness. More than 20 ALS associated genes have been identified and these genes affect distinct cellular pathways including RNA processing, nuclear protein transport, and the unfolded protein response (UPR). One of the key pathological findings is the presence of TARDBP-positive protein aggregates in the cytoplasm of neurons in the brains and spinal cords of patients. Accumulation of protein aggregates in the ER induces a UPR, which attenuates protein translation and promotes proteasome-mediated degradation as well as expression of numerous ER chaperones. Several ALS-causing genes, including VAPB, VCP and UBQLN2, have been documented to play an important role in the ER, and the loss of these proteins promotes the UPR. In addition, ER stress has also been documented in SOD1G93A heterozygous mice. Whether ER stress is toxic or protective is still a matter of debate as some data argue that ER stress may be beneficial whereas other data dispute this. If the observed ER stress is protective, other defects may accelerate the demise of neurons given that defects in proteostasis are tightly linked to ALS (Mao, 2019).

Two major pathways regulate protein clearance: proteasome and autophagy-lysosome mediated degradation. Basal autophagy is required to maintain neuronal function, as loss of autophagy has been shown to induce neurodegeneration. Emerging evidence indicates that 2 genes associated with ALS, including TARDBP and C9orf72, play a role in autophagy but how they achieve this is not well defined (Mao, 2019).

Various mutations (P56S, T46I, A145V, S160Δ, V234I) in the gene encoding the human VAPB protein cause ALS8 (OMIM: 608627), a form of ALS and spinal muscular atrophy. Interestingly, mRNAs of VAPB are decreased in sporadic patients and in neurons derived from ALS8 patients as well as in human SOD1G93A transgenic mice, suggesting that VAPB may play a role in many forms of ALS. The VAPs belong to the VAMP-associated protein family and are highly conserved across species. There are 2 VAP homologs in mammals: VAPA and B (VAPA/B). However, Drosophila has a single VAP, Vap33 which corresponds to VPR-1 in C. elegans. Studies in Drosophila, C. elegans as well as mammalian cells have shown that VAPs (Vap33, VPR-1, VAPA/B) affect multiple cellular processes. These include the size and shape of neuromuscular junctions (NMJ), the presence of a UPR, the transfer of lipids from the ER to the Golgi, mitochondrial calcium homeostasis and muscle mitochondrial dynamics. VAPA and B share an N-terminal major sperm protein (MSP) domain followed by a coiled-coil domain and a C-terminal transmembrane domain that targets the protein to the ER. Previous work documented that Drosophila Vap33 functions in a cell non-autonomous manner by releasing and secreting the MSP domain (Tsuda, 2008). The MSP domain of the human VAPB is also detected in human blood and cerebrospinal fluid (CSF) and the levels of MSP in the CSF is reduced in patients with sporadic ALS, indicating that loss of MSP secretion may be associated with different forms of ALS (Mao, 2019).

In addition to the cell non-autonomous function, VAPB also functions cell autonomously in non-vesicular lipid transfer. VAP proteins directly interact with lipid transport proteins, such as OSBP (oxysterol binding protein) and COL4A3BP/CERT through a FFAT motif (2 phenylalanines in an acidic tract) to facilitate lipid transfer. Both the OSBP and COL4A3BP/CERT proteins contain a pleckstrin homology (PH) domain that interacts with PtdIns4P on the Golgi to promote membrane tethering and lipid transfer from the ER to the Golgi. The VAP-FFAT interaction is abolished in VAPBP56S, the most prevalent variant form of VAPB in ALS8 patients. This P56S variant functions as a loss-of-function mutation in some phenotypic assays and as a dominant-negative mutation as it traps endogenous wild-type VAPA and VAPB proteins in aggregates, resulting in a partial loss of function of both VAPA and VAPB. The tethering of the ER to the Golgi facilitates the transfer of PtdIns4P from the Golgi to the ER for hydrolysis and loss of VAPs affects PtdIns4P levels, including a general increase in the cytoplasm, a decrease in the Golgi, and an increase in endosomes. However, little is known about the role of PtdIns4P in the autophagic-lysosomal degradation pathway (Mao, 2019).

This study provides both in vivo and in vitro evidence that loss of VAPs impairs endo-lysosomal degradation. It was found that loss of VAPs leads to an obvious upregulation of the PtdIns4P levels in the Golgi, and a dramatic increase in the number of endosomes, lysosomes and autophagic vesicles. These compartments are defective because they do not acidify properly. Reducing the PtdIns4P levels significantly suppresses the autophagic and lysosomal defects, suggesting that the VAPs regulate autophagy-lysosomal degradation through a PtdIns4P-mediated endosomal trafficking pathway. Impairing this pathway causes a severe defect in lysosomal degradation that may play a critical role in ALS8 and other forms of ALS (Mao, 2019).

Based on the current studies, a model is proposed. VAP proteins localize to the ER and interact with lipid transfer proteins such as OSBP and COL4A3BP/CERT through their FFAT motif. The PH domains of OSBP and COL4A3BP/CERT interact with PtdIns4P anchored on the Golgi and tether the ER to the Golgi, facilitating PtdIns4P transfer from the Golgi to the ER for its hydrolysis by SACM1L. It is argued that this leads to an accumulation of PtdIns4P in the Golgi and increased production of RAB5- and RAB7-positive endosomes. These endosomes mature into lysosomes leading to an increase in the number of lysosomes with aberrant pH. These defective lysosomes affect protein degradation, and upon fusion with autophagosomes also impair autophagic degradation, resulting in an accumulation of autophagic vesicles (Mao, 2019).

The data argue that the defects in autophagic and lysosomal degradation in VAP mutant cells are due to PtdIns4P imbalance. Indeed, by reducing the PtdIns4P to more normal levels or removing one copy of the endosome proteins Rab5 or Rab7, a significant suppression of endosome and autophagy-lysosomal defects was observed in the Vap33 mutant. Modulating the PtdIns4P and endosome pathway also rescues the locomotion deficit in Vap33 mutant animals, suggesting a strategy to modify the phenotype in patients. At the root of the elevated level of PtdIns4P is the loss of ER-Golgi tethering, as promoting ER-Golgi tethering by overexpression of an OSBP that does not require VAPs significantly suppresses the motor deficit and early lethality of mutant flies (Mao, 2019).

A recent study by Gomez-Suaga (2017) argues that the function of VAPB is to inhibit autophagy by promoting ER-mitochondria tethering. The authors argue that siRNA-mediated knockdown of VAPB in HeLa cells disrupts ER-mitochondria tethering through VAPB and its interaction with RMDN3/PTPIP51. Loss of this interaction promotes autophagy and does not impair degradation. Given that this study observed dysfunctional autophagy in flies and mammalian cells upon loss of the VAPs it is argued that VAPA and VAPB are redundant and that removing VAPB alone appears insufficient to impair lysosomal degradation, but seems sufficient to promote autophagy induction (Mao, 2019).

The combined loss of function of VAPA and B may be relevant to ALS8. Indeed, the most prevalent form of the VAPB mutation found in these patients is the P56S mutation, which functions as a dominant negative allele in some contexts and traps both VAPA and VAPB in aggregates. Hence, reducing VAPA and B may better mimic the conditions of patients with the VAPBP56S mutation. This interpretation is also consistent with the observed accumulation of SQSTM1 in aged heterozygous VAPBP56S knockin mice (Mao, 2019).

The accumulation of lumenal tagged LAMP1-GFP argues that there is a defect in lysosomal acidification upon loss of Vap33. This phenotype needs to be reconciled with the increased LysoTracker Red staining and increased Magic Red CtsB1 staining observed in Vap33 mutant cells. LysoTracker Red is activated at pH = 6.5, a higher pH than what is required to quench GFP, which is 4.5. The lysosomal pH typically ranges from 4.5 to 5.0. Hence, LysoTracker Red should label lysosomes with a pH between 4.5 ~ 6.5, including many that may not be fully functional when VAPs are lost. Similarly, CTSB has been shown to have high proteolytic activity at pH>5. Hence, LAMP1-GFP reveals non-acidified lysosomes, whereas the increased LysoTracker Red and Magic Red CtsB1 staining indicate an expansion of lysosomes that may include acidified as well as poorly acidified lysosomes. However, the current data cannot exclude the possibility that loss of VAPs impairs the trafficking of some lysosomal proteins that are trapped in non-acidic endosomes. This trafficking defect would also result in dysfunctional lysosomes, consistent with the model (Mao, 2019).

The data show that there is an increase in the acidified lysosomal pool when VAPs are lost. Interestingly, lysosomal expansion is also observed in lysosomal storage diseases due to defects in lysosomal degradation. Indeed, lysosomal degradation defects impair the processing of cargo as well as the renewal of the lysosomal compartment, leading to the accumulation of aberrant lysosomes. Furthermore, loss of VAPs results in a significant disruption of the balance of the various hydrolases per lysosome, and are consistent with the lysosomal phenotypes observed in lysosomal storage diseases (Mao, 2019).

The importance of autophagic and lysosomal function in ALS has only recently come into focus. Loss of TARDBP was recently reported to elevate the levels of TFEB and impair the fusion of autophagosomes with lysosomes. Conversely, C9orf72, the most prevalent ALS-causing gene, has been shown to decrease autophagic flux upon its loss, whereas others have argued that loss of C9orf72 promotes autophagy. However, these studies were not performed in cells that carry the G4C2 hexanucleotide expanded repeat, and the role of autophagy in C9orf72 ALS patients therefore remains to be established. The current data in flies and human cells as well as the phenotypes associated with the mice carrying a single P56S mutation argue that autophagic and lysosomal degradation may be impaired in ALS8 patients and that the primary defect is due to the upregulation of PtdIns4P upon loss of the VAP-mediated anchoring of ER to Golgi (Mao, 2019).

Search PubMed for articles about Drosophila Vap-33-1

Amarilio, R., et al. (2005). Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction. J. Biol. Chem. 280: 5934-5944. PubMed ID: 15545272

Baker, A. M. Roberts, T. M. and Stewart, M. (2002). 2.6 A resolution crystal structure of helices of the motile major sperm protein (MSP) of Caenorhabditis elegans. J. Mol. Biol. 319: 491-499. PubMed ID: 12051923

Bottino, D., et al. (2002). How nematode sperm crawl. J. Cell Sci. 115: 367-384. PubMed ID: 11839788

Brickner, J. H. and Walter, P. (2004). Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol. 2: e342. PubMed ID: 15455074

Bruijn, L. I., Miller, T. M. and Cleveland, D. W. (2004). Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci. 27: 723-749. PubMed ID: 15217349

Chai, A., et al. (2008). 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. 17: 266-280. PubMed ID: 17947296

Corrigan, C., Subramanian, R. and Miller, M. A. (2005). Eph and NMDA receptors control Ca2+/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation. Development 132: 5225-5237. PubMed ID: 16267094

Dalva, M. A. et al. (2000). EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103: 945-956. PubMed ID: 11136979

Gomez-Suaga, P., Paillusson, S., Stoica, R., Noble, W., Hanger, D. P. and Miller, C. C. J. (2017). The ER-Mitochondria tethering complex VAPB-PTPIP51 regulates autophagy. Curr Biol 27(3): 371-385. PubMed ID: 28132811

Govindan, J. A. et al. (2006). Galphao/i and Galphas signaling function in parallel with the MSP/Eph receptor to control meiotic diapause in C. elegans. Curr. Biol. 16: 1257-1268. PubMed ID: 16824915

Harris, J. E. et al. (2006). Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans. Dev. Biol. 299: 105-121. PubMed ID: 16919258

Himanen, J. P., Saha, N. and Nikolov, D. B. (2007). Cell-cell signaling via Eph receptors and ephrins. Curr. Opin. Cell Biol. 19: 534-542. PubMed ID: 17928214

Kagiwada, S. and Zen, R. (2003). Role of the yeast VAP homolog, Scs2p, in INO1 expression and phospholipid metabolism. J. Biochem. (Tokyo) 133: 515-522. PubMed ID: 12761300

Kaiser, S. E. et al. (2005). Structural basis of FFAT motif-mediated ER targeting. Structure 13: 1035-1045. PubMed ID: 16004875

Kosinski, M. et al. (2005). C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes. Development 132: 3357-3369. PubMed ID: 15975936

Kuijper, S., Turner, C. J. and Adams, R. H. (2007). Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc. Med. 17: 145-151. PubMed ID: 17574121

Loewen, C. J. and Levine, T. P. (2005). A highly conserved binding site in vesicle-associated membrane protein-associated protein (VAP) for the FFAT motif of lipid-binding proteins. J. Biol. Chem. 280: 14097-14104. PubMed ID: 15668246

Mao, D., Lin, G., Tepe, B., Zuo, Z., Tan, K. L., Senturk, M., Zhang, S., Arenkiel, B. R., Sardiello, M. and Bellen, H. J. (2019). VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway. Autophagy 15(7): 1214-1233. PubMed ID: 30741620

Miller, M. A., et al. (2001). A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation. Science 291: 2144-2147. PubMed ID: 11251118

Miller, M. A., et al. (2003). An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans. Genes Dev. 17: 187-200. PubMed ID: 12533508

Nishimura, A. L., et al. (2004). A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am. J. Hum. Genet. 75: 822-831. PubMed ID: 15372378

Nishimura, Y. et al. (1999). Molecular cloning and characterization of mammalian homologues of vesicle-associated membrane protein-associated (VAMP-associated) proteins. Biochem. Biophys. Res. Commun. 254: 21-26. PubMed ID: 9920726

Pennetta, G. et al. (2002) Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron 35: 291-306. PubMed ID: 12160747

Perry, R.J. and Ridgway, N. D. (2006). Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein. Mol. Biol. Cell 17: 2604-2616. PubMed ID: 16571669

Skehel, P. A., Fabian-Fine, R. and Kandel, E. R. (2002). Mouse VAP33 is associated with the endoplasmic reticulum and microtubules. Proc. Natl. Acad. Sci. 97: 1101-1106. PubMed ID: 10655491

Soussan, L., et al. (1999). ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles. J. Cell Biol. 146: 301-311. PubMed ID: 10427086

Teuling, et al. (2007). 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. 27: 9801-9815. PubMed ID: 17804640

Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M. A. and Bellen, H. J. (2008). The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell 133(6): 963-977. PubMed ID: 18555774

Wang, P. Y., Weng, J. and Anderson, R. G. (2005). OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation. Science 307: 1472-1476. PubMed ID: 15746430

Ward, S., et al. (1988). Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans. J. Mol. Biol. 199: 1-13. PubMed ID: 3351915

Weir, M. L., Klip A. and Trimble, W. S. (1998). Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP. Biochem. J. 333: 247-251. PubMed ID: 9657962

Whitten, S. J. and Miller, M. A. (2007). The role of gap junctions in Caenorhabditis elegans oocyte maturation and fertilization. Dev. Biol. 301: 432-446. PubMed ID: 16982048

date revised: 20 December 2008

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.