Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8.     

VAMP-Associated Protein B (VAPB) Promotes Breast Tumor Growth by Modulation of Akt Activity

VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer.     

The ALS8 protein VAPB interacts with the ER–Golgi recycling protein YIF1A and regulates membrane delivery into dendrites

The vesicle‐associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER‐like inclusions in various model systems. However, the role of wild‐type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1‐interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER–Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB‐P56S is present, YIF1A is recruited to the VAPB‐P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER‐to‐Golgi transport and that missorting of YIF1A may contribute to VAPB‐associated motor neuron disease.     

CYP2E1 and Parkinson’s disease in a MPTP-induced C57BL/6 mouse model

Background

A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca²⁺ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results

To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion

Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.     

Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S)

A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAP Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAP activities.     

The ALS8-associated mutant VAPB(P56S) is resistant to proteolysis in neurons

VAMP/synaptobrevin associated proteins A and B (VAPA and VAPB), are type IV membrane proteins enriched on ER and Golgi membranes. Both VAPA and B interact with cytoplasmic lipid transport proteins and cytoskeletal elements to maintain the structure and composition of ER and Golgi membranes. Truncated forms of both proteins are present in some tissues but the functional significance of this is not clear. In rodents processing of VAPA occurs in most tissues, however, truncated forms of VAPB have only been reported in brain tissue. It is demonstrated here that the extent of VAPB processing in rat increases during postnatal development and that it is restricted to neurons. The C-terminal polypeptide generated by this cleavage reaction remains associated with cell membranes, but its subcellular distribution is distinct from the full-length protein. A mutant form of VAPB is associated with a familial form of neurodegenerative disease, amyotrophic lateral sclerosis type 8. The mutant protein, VAPB(P56S) , is resistant to truncation in primary neuronal cultures, although remains sensitive to some form of proteolysis when over-expressed in HEK293 cells. These data suggest that neuronal cells have a particular requirement for VAPB proteolysis and that reduced levels of processed polypeptides may contribute to the neurodegeneration associated with amyotrophic lateral sclerosis type 8.     

Novel splice variants of the amyotrophic lateral sclerosis-associated gene VAPB expressed in human tissues

VAPB is a highly conserved integral membrane protein that is ubiquitously expressed in all eukaryotic organisms and located within the membranes of the endoplasmic reticulum (ER). The P56S missense mutation of the VAPB protein is linked to a hereditary form of amyotrophic lateral sclerosis (ALS8), and the pathogenesis of ALS8 has remained enigmatic. We report the cloning of five novel splice variants of the human VAPB gene, all of which are expressed at the mRNA level in the human nervous system. When transfected into human HEK293 or SH-SY5Y cells, two of these variants (VAPB-2 and VAPB-4,5) were readily detectable by immunoblotting whereas two variants (VAPB-3 and VAPB-3,4) became detectable after proteasomal inhibition, a condition commonly found in neurodegenerative diseases. Interestingly, one of these novel VAPB variants, VAPB-2, co-immunoprecipitated with wt-VAPB. However, so far none of these splice variants could be detected by immunoblotting of lysates from selected human tissues, suggesting that in vivo, the proteins translated from the variant VAPB mRNAs are quickly degraded or, alternatively, the expressed proteins are below detection limit of the available antibodies. We speculate that under conditions of proteasomal inhibition, as encountered in many neurodegenerative diseases including ALS, variant VAPB proteins might accumulate in affected cells and contribute to ALS pathogenesis.     

ALS-linked P56S-VAPB, an aggregated loss-of-function mutant of VAPB, predisposes motor neurons to ER stress-related death by inducing aggregation of co-expressed wild-type VAPB

A point mutation (P56S) in the vapb gene encoding an endoplasmic reticulum (ER)-integrated membrane protein [vesicle-associated membrane protein-associated protein B (VAPB)] causes autosomal-dominant amyotrophic lateral sclerosis. In our earlier study, we showed that VAPB may be involved in the IRE1/XBP1 signaling of the unfolded protein response, an ER reaction to inhibit accumulation of unfolded/misfolded proteins, while P56S-VAPB formed insoluble aggregates and lost the ability to mediate the pathway (loss-of-function), and suggested that P56S-VAPB promoted the aggregation of co-expressed wild-type (wt)-VAPB. In this study, a yeast inositol-auxotrophy assay has confirmed that P56S-VAPB is functionally a null mutant in vivo . The interaction between P56S-VAPB and wt-VAPB takes place with a high affinity through the major sperm protein domain in addition to the interaction through the C-terminal transmembrane domain. Consequently, wt-VAPB is speculated to preferentially interact with co-expressed P56S-VAPB, leading to the recruitment of wt-VAPB into cytosolic aggregates and the attenuation of its normal function. We have also found that expression of P56S-VAPB increases the vulnerability of NSC34 motoneuronal cells to ER stress-induced death. These results lead us to hypothesize that the total loss of VAPB function in unfolded protein response, induced by one P56S mutant allele, may contribute to the development of P56S-VAPB-induced amyotrophic lateral sclerosis.     

From ER to Eph receptors: new roles for VAP fragments

Dominantly inherited mutations in an endoplasmic reticulum protein called VAPB have been found in a subset of patients with a rare familial form of amyotrophic lateral sclerosis (ALS). In this issue, Tsuda et al. (2008) identify a secreted form of VAPB that binds directly to Eph receptors inducing their activation and signaling, providing fresh insights into ALS pathogenesis, including non-neuronal aspects of this disorder.     

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.     

Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors

The VAPB/ALS8 major sperm protein domain (vMSP) is implicated in amyotrophic lateral sclerosis and spinal muscular atrophy, yet its function in the nervous system is not well understood. In Caenorhabditis elegans and Drosophila, the vMSP is cleaved from its transmembrane anchor and secreted in a cell type-specific fashion. We show that vMSPs secreted by neurons act on Lar-like protein-tyrosine phosphatase and Roundabout growth cone guidance receptors expressed in striated muscle. This signaling pathway promotes Arp2/3-dependent actin remodeling and mitochondrial localization to actin-rich muscle I-bands. C. elegans VAPB mutants have mitochondrial localization, morphology, mobility, and fission/fusion defects that are suppressed by Lar-like receptor or Arp2/3 inactivation. Hence, growth cone guidance receptor pathways that remodel the actin cytoskeleton have unanticipated effects on mitochondrial dynamics. We propose that neurons secrete vMSPs to promote striated muscle energy production and metabolism, in part through the regulation of mitochondrial localization and function. VIDEO ABSTRACT:     

VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B

Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ～20 μM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a "fuzzy complex". 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ～12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ～3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.     

Phospholipid transfer function of PTPIP51 at mitochondria‐associated ER membranes

In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria‐associated ER membranes (MAMs). Ca²⁺ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER‐anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid‐like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51–VAPB interaction is mediated by the FFAT‐like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM‐tethering function.     

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.     

ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis

Several theories on the pathomechanism of amyotrophic lateral sclerosis (ALS) have been proposed: misfolded protein aggregates, mitochondrial dysfunction, increased glutamate toxicity, increased oxidative stress, disturbance of intracellular trafficking, and so on. In parallel, a number of drugs that have been developed to alleviate the putative key pathomechanism of ALS have been under clinical trials. Unfortunately, however, almost all studies have finished unsuccessfully. This fact indicates that the key ALS pathomechanism still remains a tough enigma. Recent studies with autopsied ALS patients and studies using mutant SOD1 (mSOD1) transgenic mice have suggested that endoplasmic reticulum (ER) stress-related toxicity may be a relevant ALS pathomechanism. Levels of ER stress-related proteins were upregulated in motor neurons in the spinal cords of ALS patients. It was also shown that mSOD1, translocated to the ER, caused ER stress in neurons in the spinal cord of mSOD1 transgenic mice. We recently reported that the newly identified ALS-causative gene, vesicle-associated membrane protein-associated protein B (VAPB), plays a pivotal role in unfolded protein response (UPR), a physiological reaction against ER stress. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB, resulting in motoneuronal vulnerability to ER stress. In this review, we summarize recent advances in research on the ALS pathomechanism especially addressing the putative involvement of ER stress and UPR dysfunction.     

VPS13D interacts with VCP/p97 and negatively regulates endoplasmic reticulum–mitochondria interactions

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER–mitochondria interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting–associated protein 13D (VPS13D) in the negative regulation of ER–mitochondria MCSs. VPS13D suppression results in extensive ER–mitochondria tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in mitochondrial morphology, mitochondrial cellular distribution, and mitochondrial DNA synthesis. Together, our results suggest that VPS13D negatively regulates the ER–mitochondria MCSs, partially through its interactions with VCP/p97.     

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.     

Amyotrophic lateral sclerosis-linked mutant VAPB enhances TDP-43-induced motor neuronal toxicity

Transactive response DNA-binding protein-43 (TDP-43) has been thought to be generally involved in the pathogenesis of most amyotrophic lateral sclerosis (ALS) patients although it remains undefined how TDP-43 is involved in the ALS pathogenesis. In this study, we found that a P56S mutant of vesicle-associated membrane protein-associated protein B (VAPB), which has been identified to be a familial ALS-causative protein, potentiated the TDP-43-induced motor neuronal cell death, while wild-type VAPB conversely inhibited it. The P56S-VAPB-induced potentiation of the TDP-43-induced death was mediated by the up-regulation of Bim expression at the mRNA level and other undefined mechanisms that leads to the enhancement of Bim and Bax activity. These observations suggest that TDP-43 and P56S-VAPB may co-operate to involve the pathogenesis of ALS.