Bullous Pemphigoid-Associated Mortality Rate in a Canadian Tertiary Referral Centre.

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune blistering disorder in adults. Most individuals with BP are over the age of 60. Its worldwide incidence has been increasing owing to population aging. Observational studies published over the last 2 decades highlight the non-negligible, albeit variable overall mortality of BP patients, with reported 12-month mortality rates of 10.8% to 40.8%, and 24-month mortality rates of 20.1% to 51.0%. Data in the Canadian population are lacking. OBJECTIVES: We aimed to estimate the 12- and 24-month overall mortality rate of Canadian patients diagnosed with BP, and to identify independent risk factors adversely impacting overall survival. METHODS: A retrospective cohort study of 166 patients with a diagnosis of BP between 2010 and 2020 was carried out at Centre hospitalier de l'Université de Montréal (CHUM), a tertiary referral center in Montréal, Québec, Canada. Cumulative mortality was calculated using the Kaplan-Meier estimator, and independent prognostic factors were identified using a Cox proportional hazards regression model. RESULTS: Eighty-five patients (51.2%) in our study were female. The median age was 79.1 years old, and 80 patients (48.2%) were 80 years old or older. Mortality at 12 and 24 months in our study cohort was 16.2% (CI95% = 10.5 - 21.8) and 27.6% (CI95% = 20.5 - 34.7), respectively. In a multivariate analysis, patients who were male, 80 years old or older, and/or had a diagnosis of a major neurocognitive disorder had a poorer overall survival. CONCLUSIONS: The all-cause mortality of patients with BP in our study population compared favorably with international data reported in the literature.

Dystonin loss-of-function leads to impaired autophagosome-endolysosome pathway dynamics.

The neuronal dystonin protein (DST-a) is a large cytoskeletal linker important for integrating the various components of the cytoskeleton. Recessive Dst mutations lead to a sensory neuropathy in mice, known as dystonia musculorum (Dstdt). The disease is characterized by ataxia, autonomic disturbances, and ultimately, death, which are associated with massive degeneration of the sensory neurons in the dorsal root ganglion (DRG). Recent investigation of Dstdt sensory neurons revealed an accumulation of autophagosomes and a disruption in autophagic flux, which was believed to be due to insufficient availability of motor protein. Motor protein levels and the endolysosomal pathway were assessed in pre-symptomatic (postnatal day 5; P5) and symptomatic (P15) stage wild-type and Dstdt DRGs. Levels of mRNA encoding molecular motors were reduced, although no significant reduction in the protein level was detected. An increase in lysosomal marker LAMP1 in medium-large size Dstdt-27J sensory neurons was observed, along with an accumulation of electron-light single-membraned vesicles in Dstdt-27J DRG tissue at the late stages of disease. These vesicles are likely to have been autolysosomes, and their presence in only late-stage Dstdt-27J sensory neurons is suggestive of a pathological defect in autophagy. Further investigation is necessary to confirm vesicle identity, and to determine the role of Dst-a in normal autophagic flux.

Epidermolysis bullosa simplex due to bi-allelic DST mutations: Case series and review of the literature.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is a heterogeneous group of inherited disorders characterized by skin fragility due to intraepidermal separation. Most cases result from heterozygous mutations in KRT5 or KRT14; however, a minority of affected individuals carry mutations in non-keratin genes including DST encoding an epithelial isoform of dystonin. DST-associated EBS is transmitted as an autosomal recessive trait. Here, we report a series of EBS patients carrying bi-allelic DST mutations and review previously reported cases aiming to delineate phenotype-genotype correlations. METHODS: Whole-exome and direct sequencing were used for variant analysis. Review of previously reported cases was performed. RESULTS: Mutation analysis revealed DST mutations in five patients belonging to three families. Two variants have not been previously reported: c.7097dupA (p.Tyr2366X) and c.7429delC (p.Leu2477Serfs*13). We identified an additional six cases in the literature, bringing the total number of individuals affected with EBS due to DST variants to 11. Patients displayed distinctive phenotypes regardless of the causative variant. CONCLUSIONS: The current study expands the clinical and genetic spectrum of DST-associated EBS subtype.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation.

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Disruption of dystonin in Schwann cells results in late-onset neuropathy and sensory ataxia.

Dystonin (Dst) is a causative gene for Dystonia musculorum (dt) mice, which is an inherited disorder exhibiting dystonia-like movement and ataxia with sensory degeneration. Dst is expressed in a variety of tissues, including the central nervous system and the peripheral nervous system (PNS), muscles, and skin. However, the Dst-expressing cell type(s) for dt phenotypes have not been well characterized. To address the questions whether the disruption of Dst in Schwann cells induces movement disorders and how much impact does it have on dt phenotypes, we generated Dst conditional knockout (cKO) mice using P0-Cre transgenic mice and Dst gene trap mice. First, we assessed the P0-Cre transgene-dependent Cre recombination using tdTomato reporter mice and then confirmed the preferential tdTomato expression in Schwann cells. In the Dst cKO mice, Dst mRNA expression was significantly decreased in Schwann cells, but it was intact in most of the sensory neurons in the dorsal root ganglion. Next, we analyzed the phenotype of Dst cKO mice. They exhibited a normal motor phenotype during juvenile periods, and thereafter, started exhibiting an ataxia. Behavioral tests and electrophysiological analyses demonstrated impaired motor abilities and slowed motor nerve conduction velocity in Dst cKO mice, but these mice did not manifest dystonic movements. Electron microscopic observation of the PNS of Dst cKO mice revealed significant numbers of hypomyelinated axons and numerous infiltrating macrophages engulfing myelin debris. These results indicate that Dst is important for normal PNS myelin organization and Dst disruption in Schwann cells induces late-onset neuropathy and sensory ataxia. MAIN POINTS: Dystonin (Dst) disruption in Schwann cells results in late-onset neuropathy and sensory ataxia. Dst in Schwann cells is important for normal myelin organization in the peripheral nervous system.

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.

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.

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).

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

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.

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.

Bullous pemphigoid (BP) patients with selective IgG autoreactivity against BP230: Review of a rare but valuable cohort with impact on the comprehension of the pathogenesis of BP.

Bullous pemphigoid (BP) is the most common autoimmune blistering disease. BP is characterized by the development of tense blisters induced by tissue-bound specific autoantibodies directed against the major autoantigens bullous pemphigoid autoantigen 180 (BP180, also called BPAG2 or Collagen XVII) and bullous pemphigoid autoantigen 230 (BP230, also called BPAG1 or dystonin). The vast majority of BP patients have autoantibodies targeting BP180, or both, BP180 and BP230. The hemidesmosomal protein BP180 is regarded as the main autoantigen, whereas the pathophysiologic relevance of intracellularly-located BP230 is controversial. A small subpopulation of BP patients selectively reveals autoantibodies against BP230 (BP230+ patients) strongly supporting that BP230 autoantibodies might be sufficient to induce skin pathology. In line, BP animal models have been developed, which successfully mimic a human BP phenotype by targeting BP230. In this context, our group has recently shown that a murine autoantibody targeting BP230 induces subepidermal blisters in vivo. This finding suggests that blister formation in the population of patients with selective autoreactivity against BP230 may share pathophysiologic features of pathogenic anti-BP230 autoantibodies in our murine model. This review summarizes the clinical features of BP patients with selective autoreactivity against BP230, enlightens the currently available BP mouse models targeting BP230 and discusses the potential pathophysiological mechanism of BP230 autoantibodies.

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.

Characterization of gastrointestinal pathologies in the dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

BACKGROUND: Dystonia musculorum (Dstdt ) is a murine disease caused by recessive mutations in the dystonin (Dst) gene. Loss of dorsal root ganglion (DRG) sensory neurons, ataxia, and dystonic postures before death by postnatal day 18 (P18) is a hallmark feature. Recently we observed gas accumulation and discoloration in the small intestine and cecum in Dstdt mice by P15. The human disease resulting from dystonin loss-of-function, known as hereditary sensory and autonomic neuropathy type VI (HSAN-VI), has also been associated with gastrointestinal (GI) symptoms including chronic diarrhea and abdominal pain. As neuronal dystonin isoforms are expressed in the GI tract, we hypothesized that dystonin loss-of-function in Dstdt-27J enteric nervous system (ENS) neurons resulted in neurodegeneration associated with the GI abnormalities. METHODS: We characterized the nature of the GI abnormalities observed in Dstdt mice through histological analysis of the gut, assessing the ENS for signs of neurodegeneration, evaluation of GI motility and absorption, and by profiling the microbiome. KEY RESULTS: Though gut histology, ENS viability, and GI absorption were normal, slowed GI motility, thinning of the colon mucous layer, and reduced microbial richness/evenness were apparent in Dstdt-27J mice by P15. Parasympathetic GI input showed signs of neurodegeneration, while sympathetic did not. CONCLUSIONS & INFERENCES: Dstdt-27J GI defects are not linked to ENS neurodegeneration, but are likely a result of an imbalance in autonomic control over the gut. Further characterization of HSAN-VI patient GI symptoms is necessary to determine potential treatments targeting symptom relief.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

Functional and Genetic Analysis of Neuronal Isoforms of BPAG1.

The neuronal isoforms of bullous pemphigoid antigen 1 (BPAG1, and also known as dystonin) are a group of large cytoskeletal linker proteins predominantly expressed in sensory neurons. The major neuronal isoforms consist of the spectraplakins (BPAG1/dystonin-a1, -a2, -a3), which have an N-terminus actin-binding domain and a C-terminus microtubule-binding domain. These proteins have crucial roles in cytoskeletal organization and stability, organelle integrity, and intracellular transport. BPAG1 loss-of-function in mice results in a lethal movement disorder known as dystonia musculorum (dt), which is likely caused by rapid sensory neuron degeneration. A human disease termed hereditary and sensory autonomic neuropathy type VI was also identified to be associated with mutations in the BPAG1 gene (DST). This chapter provides an overview of the type of experiments used for analysis of the different isoforms of BPAG1.

Disruption in the autophagic process underlies the sensory neuropathy in dystonia musculorum mice.

A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst(dt)) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst(dt-Tg4) mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst(dt-Tg4) sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst(dt-Tg4) mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst(dt-Tg4) mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst(dt-Tg4) dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst(dt) pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.