Roles of dystonin isoforms in the maintenance of neural, muscle, and cutaneous tissues.

Dystonin (DST), also known as bullous pemphigoid antigen 1 (BPAG1), encodes cytoskeletal linker proteins belonging to the plakin family. The DST gene produces several isoforms, including DST-a, DST-b, and DST-e, which are expressed in neural, muscle, and cutaneous tissues, respectively. Pathogenic DST mutations cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) and epidermolysis bullosa simplex (EBS); therefore, it is important to elucidate the roles of DST isoforms in multiple organs. Recently, we have used several Dst mutant mouse strains, in which the expression of Dst isoforms is disrupted in distinct patterns, to gain new insight into how DST functions in multiple tissues. This review provides an overview of the roles played by tissue-specific DST isoforms in neural, muscle, and cutaneous tissues.

Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum.

The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.

Isoform-specific mutation in Dystonin-b gene causes late-onset protein aggregate myopathy and cardiomyopathy.

Dystonin (DST), which encodes cytoskeletal linker proteins, expresses three tissue-selective isoforms: neural DST-a, muscular DST-b, and epithelial DST-e. DST mutations cause different disorders, including hereditary sensory and autonomic neuropathy 6 (HSAN-VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-related diseases has been unclear. Because DST-b contains all of the DST-a-encoding exons, known HSAN-VI mutations could affect both DST-a and DST-b isoforms. To investigate the specific function of DST-b in striated muscles, we generated a Dst-b-specific mutant mouse model harboring a nonsense mutation. Dst-b mutant mice exhibited late-onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also found nuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations as a unique pathological hallmark in Dst-b mutation-induced cardiomyopathy. RNA-sequencing analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico analysis identified DST-b alleles with nonsense mutations in populations worldwide, suggesting that some unidentified hereditary myopathy and cardiomyopathy are caused by DST-b mutations. Here, we demonstrate that the Dst-b isoform is essential for long-term maintenance of striated muscles.

Diverse dystonin gene mutations cause distinct patterns of Dst isoform deficiency and phenotypic heterogeneity in Dystonia musculorum mice.

Loss-of-function mutations in dystonin (DST) can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, DST-related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single DST locus produces at least three major DST isoforms: DST-a (neuronal isoform), DST-b (muscular isoform) and DST-e (epithelial isoform). Dystonia musculorum (dt) mice, which have mutations in Dst, were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of DST-related diseases, we investigated two mutant strains with different mutations: a spontaneous Dst mutant (Dstdt-23Rbrc mice) and a gene-trap mutant (DstGt mice). The Dstdt-23Rbrc allele possesses a nonsense mutation in an exon shared by all Dst isoforms. The DstGt allele is predicted to inactivate Dst-a and Dst-b isoforms but not Dst-e There was a decrease in the levels of Dst-a mRNA in the neural tissue of both Dstdt-23Rbrc and DstGt homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both Dstdt-23Rbrc and DstGt mice at postnatal stages. In contrast, Dst-e mRNA expression was reduced in the skin of Dstdt-23Rbrc mice but not in DstGt mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with Dst mRNAs. Because Dst-e encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of Dstdt-23Rbrc mice but not in those of DstGt mice; thus, the distinct phenotype of the skin of Dstdt-23Rbrc mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the Dst locus can cause different loss-of-function patterns among Dst isoforms, which accounts for the heterogeneous neural and skin phenotypes in dt mice and DST-related diseases.

Role of BP230 autoantibodies in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune disease associated with subepidermal blistering due to autoantibodies directed against BP180 and BP230. BP180 is currently considered as the major pathogenic autoantigen. However, previous clinical findings suggested that anti-BP230 autoantibodies alone can cause skin lesions in animal models and many BP patients. The characteristics of BP230 and the pathogenic roles of anti-BP230 antibodies have been proposed. First, at the molecular level, BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque and interacts with other constituents of hemidesmosomes. Second, the presence of BP230 autoantibodies may correlate with specific clinical features of BP. The immunoglobulin (Ig)G autoantibodies from BP patients react mainly against the C-terminus of BP230, while the IgE autoantibodies are still inconclusive. Third, in vivo, autoantibodies against BP230 involved in the disease may not only induce the inflammatory response but also impair the structural stability of hemidesmosomes. This article reviews recently published work about the role of BP230 and its antibodies, including IgG and IgE, aiming to find clues of its clinical association and lay the foundation for the research on the pathogenicity of antibodies against BP230.

The Barrier Molecules Junction Plakoglobin, Filaggrin, and Dystonin Play Roles in Melanoma Growth and Angiogenesis.

OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Integrin α6ß4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.

Dystonin/BPAG1 modulates diabetes and Alzheimer's disease cross-talk: a meta-analysis.

Dementia is one of the diabetic complications under intensive study. Alteration of synaptic adhesion protein (SAP) associates with neurological diseases, including Alzheimer's disease. However, the regulation of SAPs in the brain of diabetes mellitus remains elusive. To pinpoint the candidate SAPs underlining the mechanism of diabetic dementia, we investigated expression profiling of SAPs in both streptozotocin (STZ)-induced diabetic mice, AppNL-G-F/NL-G-F mice, and amyloid precursor protein intracellular domain (AICD)-induced human neural cell line from public databases. DST (Dystonin/BPAG1) was identified upregulated in both models. Our finding suggests that DST alteration may involve in the mechanism of diabetic dementia.

Anti-BP180 Autoantibodies Are Present in Stroke and Recognize Human Cutaneous BP180 and BP180-NC16A.

Objective: Current evidence has revealed a significant association between bullous pemphigoid (BP) and neurological diseases (ND), including stroke, but the incidence of BP autoantibodies in patients with stroke has not previously been investigated. Our study aimed to assess BP antigen-specific antibodies in stroke patients. Design: One hundred patients with stroke and 100 matched healthy controls were randomly selected for measurement of anti-BP180/BP230 IgG autoantibodies by enzyme-linked immunosorbent assay (ELISA), salt-split indirect immunofluorescence (IIF), and immunoblotting against human cutaneous BP180 and BP180-NC16A. Results: Anti-BP180 autoantibodies were found in 14 (14.0%) patients with stroke and 5 (5.0%) of controls by ELISA (p < 0.05). Sera from 13 (13.0%) patients with stroke and 3 (3.0%) controls reacted with 180-kDa proteins from human epidermal extract (p < 0.05). 11 (11.0%) of stroke and 2 (2.0%) of control sera recognized the human recombinant full length BP180 and NC16A (p < 0.05). The anti-BP180-positive patients were significantly younger than the negative patients at the time of stroke (p < 0.001). Conclusion: Development of anti-BP180 autoantibodies occurs at a higher frequency after stroke, suggesting BP180 as a relatively common autoantigen after stroke and providing novel insights into BP pathogenesis in aging.

Biological predictors shared by dementia and bullous pemphigoid patients point out a cross-antigenicity between BP180/BP230 brain and skin isoforms.

Bullous pemphigoid (BP) following dementia diagnosis has been reported in the elderly. Skin and brain tissues express BP180 and BP230 isoforms. Dementia has been associated with rs6265 (Val66Met) polymorphism of the brain-derived neurotrophic factor (BDNF) gene and low serum BDNF. Here we investigated a possible cross-antigenicity between BP180/BP230 brain and skin isoforms. We assessed antibodies against BP180/BP230 and BDNF levels by ELISA and BDNF Val66Met SNP by PCR in three groups: 50 BP patients, 50 patients with dementia, and 50 elderly controls. Heatmap hierarchical clustering and data mining decision tree were used to analyze the patients' demographic and laboratorial data as predictors of dementia-BP association. Sixteen percent of BP patients with the lowest serological BDNF presented dementia-BP clinical association. Anti-BP180/230 positivity was unexpected observed among dementia patients (10%, 10%) and controls (14%, 1%). Indirect immunofluorescence using healthy human skin showed a BP pattern in two of 10 samples containing antibodies against BP180/BP230 obtained from dementia group but not in the control samples. Neither allelic nor genotypic BDNF Val66Met SNP was associated with dementia or with BP (associated or not with clinical manifestation of dementia). Heatmap analysis was able to differentiate the three studied groups and confirmed the ELISA results. The comprehensive data mining analysis revealed that BP patients and dementia patients shared biological predictors that justified the dementia-BP association. Autoantibodies against the BP180/BP230 brain isoforms produced by dementia patients could cross-react with the BP180/BP230 skin isoforms, which could justify cases of dementia preceding the BP disease.

Dystonin-A3 upregulation is responsible for maintenance of tubulin acetylation in a less severe dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

Hereditary sensory and autonomic neuropathy type VI (HSAN-VI) is a recessive human disease that arises from mutations in the dystonin gene (DST; also known as Bullous pemphigoid antigen 1 gene). A milder form of HSAN-VI was recently described, resulting from loss of a single dystonin isoform (DST-A2). Similarly, mutations in the mouse dystonin gene (Dst) result in severe sensory neuropathy, dystonia musculorum (Dstdt). Two Dstdt alleles, Dstdt-Tg4 and Dstdt-27J, differ in the severity of disease. The less severe Dstdt-Tg4 mice have disrupted expression of Dst-A1 and -A2 isoforms, while the more severe Dstdt-27J allele affects Dst-A1, -A2 and -A3 isoforms. As dystonin is a cytoskeletal-linker protein, we evaluated microtubule network integrity within sensory neurons from Dstdt-Tg4 and Dstdt-27J mice. There is a significant reduction in tubulin acetylation in Dstdt-27J indicative of microtubule instability and severe microtubule disorganization within sensory axons. However, Dstdt-Tg4 mice have no change in tubulin acetylation, and microtubule organization was only mildly impaired. Thus, microtubule instability is not central to initiation of Dstdt pathogenesis, though it may contribute to disease severity. Maintenance of microtubule stability in Dstdt-Tg4 dorsal root ganglia could be attributed to an upregulation in Dst-A3 expression as a compensation for the absence of Dst-A1 and -A2 in Dstdt-Tg4 sensory neurons. Indeed, knockdown of Dst-A3 in these neurons resulted in a decrease in tubulin acetylation. These findings shed light on the possible compensatory role of dystonin isoforms within HSAN-VI, which might explain the heterogeneity in symptoms within the reported forms of the disease.

BPAG1 in muscles: Structure and function in skeletal, cardiac and smooth muscle.

BPAG1, also known as Dystonin or BP230, belongs to the plakin family of proteins, which has multiple cytoskeleton-binding domains. Several BPAG1 isoforms are produced by a single BPAG1 genomic locus using different promoters and exons. For example, BPAG1a, BPAG1b, and BPAG1e are predominantly expressed in the nervous system, muscle, and skin, respectively. Among BPAG1 isoforms, BPAG1e is well studied because it was first identified as an autoantigen in patients with bullous pemphigoid, an autoimmune skin disease. BPAG1e is a component of hemidesmosomes, the adhesion complexes that promote dermal-epidermal cohesion. In the nervous system, the role of BPAG1a is also well studied because disruption of BPAG1a results in a phenotype identical to that of Dystonia musculorum (dt) mutants, which show progressive motor disorder. However, the expression and function of BPAG1 in muscles is not well studied. The aim of this review is to provide an overview of and highlight some recent findings on the expression and function of BPAG1 in muscles, which can assist future studies designed to delineate the role and regulation of BPAG1 in the dt mouse phenotype and in human hereditary sensory and autonomic neuropathy type 6 (HSAN6).

BPAG1, a distinctive role in skin and neurological diseases.

Spectraplakins are multifunctional cytoskeletal linker proteins that act as important communicators, connecting cytoskeletal components with each other and to cellular junctions. Bullous pemphigoid antigen 1 (BPAG1)/dystonin is a member of spectraplakin family and expressed in various tissues. Alternative splicing of BPAG1 gene produces various isoforms with unique structure and domains. BPAG1 plays crucial roles in numerous biological processes, such as cytoskeleton organization, cell polarization, cell adhesion, and cell migration as well as signaling transduction. Genetic mutation of BPAG1 isoforms is the miscreant of epidermolysis bullosa and multifarious, destructive neurological diseases. In this review, we summarize the recent advances of BPAG1's role in various biological processes and in skin and neurological diseases.

Novel mutations in dystonin provide clues to the pathomechanisms of HSAN-VI.

OBJECTIVE: To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (DST) gene and to evaluate their effect on neurons derived from induced pluripotent stem cells (iPSC). METHODS: The family consisted of 3 affected siblings from nonconsanguineous healthy parents. All members underwent clinical and electrophysiologic evaluation and genetic analysis. Two patients underwent quantitative sensory testing (QST), cardiovascular reflexes, dynamic sweat test, and skin biopsy to evaluate somatic and autonomic cutaneous innervation and to get fibroblast cultures for developing iPSC-derived neurons. RESULTS: Onset occurred in the first decade, with painless and progressive mutilating distal ulcerations leading to amputation and joint deformity. Sensation to pain, touch, and vibration was reduced. Autonomic disturbances included hypohidrosis, pupillary abnormalities, and gastrointestinal and sexual dysfunction. Nerve conduction studies showed a severe axonal sensory neuropathy. QST and autonomic functional studies were abnormal. Skin biopsy revealed a lack of sensory and autonomic nerve fibers. Genetic analysis revealed 2 pathogenic mutations in the DST gene affecting exclusively the DST neuronal isoform-a2. Neurons derived from iPSC showed absence or very low levels of DST protein and short and dystrophic neuritis or no projections at all. CONCLUSIONS: Unlike the previous HSAN-VI family, our description indicates that DST mutations may be associated with a nonlethal and nonsyndromic phenotype. Neuronal loss affects large and small sensory nerve fibers as well as autonomic ones. Induced-PSC findings suggest that dystonin defect might alter proper development of the peripheral nerves. Dystonin-a2 plays a major role in the HSAN-VI phenotype.

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality.

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (DstGt) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt23Rbrc. Motor abnormalities in homozygous dt23Rbrc/dt23Rbrc mice are not as severe as homozygous DstGt/DstGt mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt23Rbrc/dt23Rbrc mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt23Rbrc/dt23Rbrc mice than in homozygous DstGt/DstGt mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.

Axonopathy in the Central Nervous System Is the Hallmark of Mice with a Novel Intragenic Null Mutation of Dystonin.

Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary sensory autonomic neuropathy. Mutated mice show severe movement disorders and die at the age of 3-4 weeks. This study describes the discovery and molecular, clinical, as well as pathological characterization of a new spontaneously occurring mutation in the dystonin gene in C57BL/6N mice. The mutation represents a 40-kb intragenic deletion allele of the dystonin gene on chromosome 1 with exactly defined deletion borders. It was demonstrated by Western blot, mass spectrometry, and immunohistology that mice with a homozygous mutation were entirely devoid of the dystonin protein. Pathomorphological lesions were restricted to the brain stem and spinal cord and consisted of swollen, argyrophilic axons and dilated myelin sheaths in the white matter and, less frequently, total chromatolysis of neurons in the gray matter. Axonal damage was detected by amyloid precursor protein and nonphosphorylated neurofilament immunohistology. Axonopathy in the central nervous system (CNS) represents the hallmark of this disease. Mice with the dystonin mutation also showed suppurative inflammation in the respiratory tract, presumably due to brain stem lesion-associated food aspiration, whereas skeletal muscles showed no pathomorphological changes. This study describes a novel mutation in the dystonin gene in mice leading to axonopathy in the CNS. In further studies, this model may provide new insights into the pathogenesis of neurodegenerative diseases and may elucidate the complex interactions of dystonin with various other cellular proteins especially in the CNS.

One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.