Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

The transcription factor NF-YA is crucial for neural progenitor maintenance during brain development.

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.

GPATCH4 contributes to nucleolus morphology and its dysfunction impairs cell viability.

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated ß-galactosidase (SA-ß-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability.

Modeling familial and sporadic Parkinson's disease in small fishes.

The establishment of animal models for Parkinson's disease (PD) has been challenging. Nevertheless, once established, they will serve as valuable tools for elucidating the causes and pathogenesis of PD, as well as for developing new strategies for its treatment. Following the recent discovery of a series of PD causative genes in familial cases, teleost fishes, including zebrafish and medaka, have often been used to establish genetic PD models because of their ease of breeding and gene manipulation, as well as the high conservation of gene orthologs. Some of the fish lines can recapitulate PD phenotypes, which are often more pronounced than those in rodent genetic models. In addition, a new experimental teleost fish, turquoise killifish, can be used as a sporadic PD model, because it spontaneously manifests age-dependent PD phenotypes. Several PD fish models have already made significant contributions to the discovery of novel PD pathological features, such as cytosolic leakage of mitochondrial DNA and pathogenic phosphorylation in α-synuclein. Therefore, utilizing various PD fish models with distinct degenerative phenotypes will be an effective strategy for identifying emerging facets of PD pathogenesis and therapeutic modalities.

Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP.

Golli-myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli-myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli-myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli-myelin basic protein knockout through the generation of conditional knockout mice (Golli-myelin basic proteinsfl/fl; E3CreN), in which Golli-myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli-myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli-myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli-myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli-myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli-myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.

FACS-array-based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model.

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis.

Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study.

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates.

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

Reappraisal of VAChT-Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers.

VAChT-Cre.Fast and VAChT-Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT-Cre lines with a reporter line, CAG-Syp/tdTomato, in which synaptophysin-tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co-localized with osteopontin, a recently discovered motor neuron marker for slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) types. The fluorescence did not preferentially co-localize with matrix metalloproteinase-9, a marker for fast-twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT-Cre lines are Cre-drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT-Cre.Fast and VAChT-Cre.Slow to VAChT-Cre.Early and VAChT-Cre.Late, respectively. The mouse lines will be useful tools to study slow-type motor neurons, in relation to physiology and pathology.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ µM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy.

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.

Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor.

In Huntington's disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF-Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF-Y binding to the promoter region of HSP70, one of the NF-Y targets. RT-PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF-Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF-Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF-Y could be an important target of mutant Huntingtin.

Proteomic analysis of heat-stable proteins revealed an increased proportion of proteins with compositionally biased regions.

Intrinsically disordered proteins (IDPs) have been in the spotlight for their unique properties, such as their lack of secondary structures and low sequence complexity. Alpha-synuclein and tau are representative disease-related IDPs with low complexity regions in their sequences, accumulating in the brains of patients with Parkinson disease and Alzheimer disease, respectively. Their heat resistance in particular was what attracted our attention. We assumed that there exist many other unidentified proteins that are resistant to heat-treatment, referred to as heat-stable proteins, which would also have low sequence complexity. In this study, we performed proteomic analysis of heat-stable proteins of mouse brains and found that proteins with compositionally biased regions are abundant in the heat-stable proteins. The proteins related to neurodegeneration are known to undergo different types of post-translational modifications (PTMs) such as phosphorylation and ubiquitination. We then investigated the heat-stability and aggregation properties of phosphorylated synuclein and tau with different phosphorylation sites. We suggest that PTMs can be important factors that determine the heat-stability and aggregation properties of a protein. IDPs identified in the heat-stable proteins of mouse brains would be candidates for the pathogenic proteins for neurodegeneration.

Amyloids facilitate DNA transfection in vivo.

Amyloid fibril deposits are a main source of pathology in neurodegenerative diseases. Normal proteins such as tau, alpha-synuclein, TDP-43 and others could form specific conformational fibrils called amyloid, which deposited in the brains of neurodegenerative diseases. Although the pathological roles of amyloids in cell death have been discussed a lot, their other functions have not been investigated well. Here, we studied the effect of amyloids on DNA transfection in vivo. We injected quantum dot labeled or non-labeled amyloid-preformed fibrils (PFFs) and a green fluorescent protein (EGFP) expression vector into organs including brain, testis, liver and calf muscle. GFP expression patterns were examined by immunohistochemistry and western blotting. At 24 h after injection, EGFP was predominantly expressed in the neurons in the cortex and the striatum, Leydig cells in testis, hepatocytes in the liver and muscle cells. EGFP expression was inhibited by an endocytosis inhibitor, sertraline in the brain and testis. The amyloid-PFFs potentiated Ca2+ transients shown by calcium imaging and EGFP expression in the brain was blocked by Ca blocker, cilnidipine. Our results show that amyloid-PFFs facilitate DNA transfection and can be used for a new gene delivery system in vivo.

Hornerin deposits in neuronal intranuclear inclusion disease: direct identification of proteins with compositionally biased regions in inclusions.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.

Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis?

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

Proteomic analysis of subcellular compartments containing disseminated alpha-synuclein seeds.

The pathological form of a-synuclein (a-syn) is transmitted through neural circuits in the brains of Parkinson disease (PD) patients and amplifies misfolded a-syn, further forming intracellular deposits. However, the details of a-syn pre-formed fibrils (PFFs) transmission in vivo have not been fully elucidated. By inoculating Quantum dots (QD)-labeled a-syn PFFs (QD-a-syn PFFs) into the unilateral striatum, we detected QD-a-syn PFFs in brain homogenates obtained from the ipsilateral and contralateral sides of the inoculated site and further obtained QD-a-syn PFFs enriched-particles with fluorescence-activated organelle sorting. Proteomic analysis suggested that QD-a-syn PFFs-enriched particles in the contralateral side were associated with component proteins of synapse. In contrast, QD-a-syn PFFs-enriched particles in the ipsilateral side were associated with proteins belonging to ER components. Immunostaining of brain sections confirmed that QD-a-syn PFFs in the contralateral side were co-localized with synaptic vesicle marker proteins in the cortex and striatum. Additionally, QD-a-syn PFFs in the ipsilateral side were more co-localized with ER marker proteins compared to the contralateral side. These results correspond to proteomic analysis. This study provides potential candidates for the subcellular localization of a-syn PFFs in vivo during the dissemination phase of seeds. These subcellular compartments could be involved in the transmission of seeds.

Intracellular polarity protein PAR-1 regulates extracellular laminin assembly by regulating the dystroglycan complex.

Cell polarity depends on extrinsic spatial cues and intrinsic polarity proteins including PAR-aPKC proteins. In mammalian epithelial cells, cell-cell contacts provide spatial cues that activate the aPKC-PAR-3-PAR-6 complex to establish the landmark of the initial cellular asymmetry. PAR-1, a downstream target of the aPKC-PAR-3-PAR-6 complex, mediates further development of the apical and basolateral membrane domains. However, the relationships between the PAR-aPKC proteins and other extrinsic spatial cues provided by the extracellular matrix (ECM) remain unclear. Here, we show that PAR-1 colocalizes with laminin receptors and is required for the assembly of extracellular laminin on the basal surface of epithelial cells. Furthermore, PAR-1 regulates the basolateral localization of the dystroglycan (DG) complex, one of the laminin receptors essential for basement membrane formation. We also show that PAR-1 interacts with the DG complex and is required for the formation of a functional DG complex. These results reveal the presence of a novel inside-out pathway in which an intracellular polarity protein regulates the ECM organization required for epithelial cell polarity and tissue morphogenesis.

Proteomics-Based Approach Identifies Altered ER Domain Properties by ALS-Linked VAPB Mutation.

An ER transmembrane protein, vesicle-associated membrane protein-associated protein B (VAPB), binds to several organelle-resident membrane proteins to mediate ER-organelle tethering. Mutation in amyotrophic lateral sclerosis (ALS) induces protein misfolding and aggregation, leading to ER disorganization. Gain or loss of function is suggested for VAPB mutation, however comprehensive study focusing on VAPB-ER domain has yet been performed. We here conducted proteomic characterization of the ER containing VAPB and its ALS-linked P56S mutant. For this purpose, we first optimized the proteomics of different ER domains immuno-isolated from cultured cells, and identified ER sheet- and tubule-specific proteomes. By using these as references, we found that VAPB-ER proteome had intermediate ER domain properties but its tubular property was specifically decreased by its mutation. Biochemical, immunofluorescence and proximity ligation assays suggested this was mediated by delocalization of VAPB from ER tubules. The VAPB-ER proteomics further suggested reduced incorporation of multiple proteins located in different organelles, which was confirmed by proximity ligation assay. Taken together, our proteomics-based approach indicates altered ER domain properties and impaired ER-organelle tethering by VAPB mutation.