Dystonin Is Essential for Maintaining Neuronal Cytoskeleton Organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons-cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology. Copyright 1998 Academic Press.

Heterogeneity of ANCA sera showing atypical, peripheral and classical cytoplasmic immunofluorescence patterns.

Atypical (A) ANCA immunofluorescence (IF) patterns have been described in several disease groups. We have previously reported a distinct cytoplasmic A-ANCA in 7-10% of patients with SLE and/or RA. Here, we show that these rheumatic disease associated A-ANCAs are best identified using U937 cells as substrate and that they do not target either a serine proteinase or a peroxidase. Furthermore, these sera immunoprecipitate a 40 kDa or a 42 kDa band using in vivo 35S-amino acid labelled HL60 or U937 cell extracts, respectively. Although these bands are the only one seen with pure A-ANCA sera, they can also be found in addition to the expected bands of PR3 or MPO in up to 30% of bona fide C- or P-ANCA sera. These data confirm and extend our previous observations. They also suggest that target heterogeneity of ANCA antibodies is frequent. Care should thus be taken in interpreting in a cause and effect relationship, an IF pattern or a biological effect produced by a serum with ill documented monospecificity. The exact nature and significance of this (these) new antigen(s) have yet to be clarified.

Cloning and characterization of the neural isoforms of human dystonin.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5' exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical.

Dystonin transcripts are altered and their levels are reduced in the mouse neurological mutant dt24J.

Dystonia musculorum is a hereditary mouse neurodegenerative disorder that primarily affects the sensory arm of the nervous system. We have recently cloned and identified a candidate gene for this disorder and designated it dystonin. The sequence of dystonin predicts a rod-shaped cytoskeletal-associated protein with an actin-binding domain at the N-terminal end and a hemidesmosomal protein sequence (bpag1) at the C-terminal end. Here we show that abnormal dystonin transcripts are present in neural tissues of a spontaneous dystonia musculorum mutant, dt24J. We further show that dystonin transcript levels are reduced 2- to 3-fold in dt24J mice.

Dystonin expression in the developing nervous system predominates in the neurons that degenerate in dystonia musculorum mutant mice.

Dystonia musculorum (dt) is an inherited neurodegenerative disorder in mice. The dt gene product, dystonin, contains the bullous pemphigoid antigen 1 coding region at its C-terminus and an actin binding domain at its N-terminus. We demonstrate that dystonin expression throughout mouse development predominates in neurons of the cranial and spinal sensory ganglia. These structures are the most severely affected in dystonic mice which could explain their severe sensory ataxia. Since we show expression in sensory neurons with small and large axoplasmic volumes, but degeneration is restricted primarily to the latter type, we suggest that caliber and size of the axon is an important factor in the disease process. Dystonin is also expressed in the extrapyramidal motor system and in the cerebellum. Functional defects in these cell types could account for the dystonic symptoms of dt mice not explained by simple sensory denervation. We also detect dystonin expression in motor neurons most of which are unaffected by the degenerative process in dt mice.

Detection of anti-neutrophil cytoplasmic antibodies by immunoprecipitation.

Immunoprecipitation (IP) of radiolabeled PMN extracts was used as the gold standard for anti-proteinase 3 (PR-3) autoantibody detection to validate immunofluorescence (IF) and alpha granule (alpha) ELISA. A myeloperoxidase (MPO) ELISA was also used in parallel. We studied 48 patients with strictly defined vasculitic syndromes in the initial active phase of their disease. The 3 methods confirmed the high (> 90%) sensitivity and specificity of anti-PR-3 for patients with Wegener's granulomatosis (WG). Similarly, a high (86%) sensitivity of MPO-ELISA was found in microscopic polyarteritis as defined. Using alpha-ELISA, we could not improve the detection rate of anti-PR-3 obtained by IF-cANCA-pattern reading. Moreover, a small proportion (< 15%) of biopsy-proven WG patients had anti-MPO antibodies detected by IF, usually as pANCA but also, even if rarely, as bona fide cANCA (< 5%). Thus, IF would seem to be the most reliable screening method and alpha-ELISA should be used for confirmation. On the other hand, because MPO-ELISA detected twice as many anti-MPO positive sera as did pANCA pattern reading by IF, we suggest that in the clinical context of a vasculitis, MPO-ELISA should also be used as a screening test. Although IP is not designed for routine clinical use, it should be required when reporting the presence of anti-PR-3 in vasculitis-like diseases that are fertile grounds for false positive reactions.

Dystonin is essential for maintaining neuronal cytoskeleton organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.

Dystonin-deficient mice exhibit an intrinsic muscle weakness and an instability of skeletal muscle cytoarchitecture.

Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.