Impaired fast axonal transport in neurons of the sciatic nerves from dystonia musculorum mice

Dystonia musculorum (dt) mice suffer from a severe sensory neuropathy caused by mutations in the gene encoding the cytoskeletal cross-linker protein dystonin/bullous pemphigoid antigen 1 (Bpag1). Loss of function of dystonin/Bpag1 within neurons leads to a loss in the maintenance of cytoskeletal organization and to the development of focal axonal swellings prior to death of the neuron. In the present study, we demonstrate that neurons within the sciatic nerves of dt27J mice undergo axonal degeneration as has been previously reported for the dorsal roots. Furthermore, ultrastructural studies reveal a perturbed organization of the neurofilament and microtubule networks within the axons of sciatic nerves in dt27J mice. The disrupted cytoskeletal organization suggested that axonal transport is affected in dt mice. To address this, we assessed fast axonal transport by measuring the rate of accumulation of acetylcholinesterase (AChE) proximal and distal to a surgically introduced ligature on the sciatic nerves of normal and dt27J mice. Our findings demonstrate that axonal transport of AChE in both orthograde and retrograde directions is markedly affected, and allow us to conclude that axonal transport defects do exist in the sciatic nerves of dt27J mice.     

Dystonin/Bpag1--a link to what?

The dystonin/Bpag1 cytoskeletal interacting proteins play important roles in maintaining cytoarchitecture integrity in skin and in the neuromuscular system. The most profound phenotype observed in the dystonin mutant dystonia musculorum (dt) mice is a severe movement disorder, attributed in large part to sensory neuron degeneration. The molecular basis for this phenotype is currently not clear, despite several studies indicating possible causes for the pathology in dt mice. Complicating the picture of what essential dystonin functions are lost in dt mice is the fact that our understanding of the very nature of what dystonin is has evolved greatly over the past decade. Elucidating the roles of dystonin most relevant to neuronal function and survival should help to shed light on some of the common mechanisms underlying neurodegeneration.     

Neuronal dystonin isoform 2 is a mediator of endoplasmic reticulum structure and function

Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt(27) mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca(2+) dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.     

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.     

Motor unit abnormalities in Dystonia musculorum mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt(27J) mice. Phenotypic dt(27J) mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt(27J) mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt(27J) mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt(27J) muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt(27J) mice. This study reveals neuromuscular defects that likely contribute to the dt(27J) pathology and identifies a critical role for dystonin outside of sensory neurons.     

Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. Although much investigation has focused on understanding dt pathology, the diverse cellular functions of dystonin isoforms remain poorly characterized. In this paper, we highlight novel functions of the dystonin-a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform-specific loss-of-function analysis, we found dystonin-a2 bound to MT-associated protein 1B (MAP1B) in the centrosomal region, where it maintained MT acetylation. In dt neurons, absence of the MAP1B-dystonin-a2 interaction resulted in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MT accumulation resulted in Golgi fragmentation and prevented anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A administration or MAP1B overexpression mitigated the observed defect. These cellular aberrations are apparent in prephenotype dorsal root ganglia and primary sensory neurons from dt mice, suggesting they are causal in the disorder.     

Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana . Twenty fast neutron-generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate-sized deletions at HY4 , while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis . Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4 .     

Differentiation potential of primary myogenic cells derived from skeletal muscle of dystonia musculorum mice

The dystonia musculorum (dt) mouse has a mutation in the gene encoding the cytoskeletal crosslinker protein bullous pemphigoid antigen 1 (Bpag1). These mice have perturbations in the cytoarchitecture of skeletal muscle. Bpag1 has been hypothesized to be involved in the maintenance rather than the establishment of the muscle cell architecture given that cytoskeletal disruptions are observed in the muscle tissue of post-natal dt mice. Not known is whether Bpag1-deficiency affects the proliferative and differentiation potential of myogenic cells. In the present investigation, we show that the growth rate of cultured primary myogenic cells derived from dt mice, as assessed by BrdU incorporation, is similar to that of myogenic cells derived from wild-type littermates. The myogenic differentiation potential of dt versus wild-type cells was monitored by examining the expression of myosin heavy chain by immunofluorescence, and by analyzing the expression profiles of myogenic regulatory factors and myogenic differentiation markers by RT-PCR. In all instances, both dt and wild-type myogenic cells displayed a similar differentiation profile. Furthermore, the absence of any observable differences in the proliferation and differentiation rates of dt and wild-type cells was not due to an overexpression of plectin, another crosslinker protein, in dt cells. Together, these findings demonstrate that the early phases of myogenic differentiation occur independently of Bpag1.     

Genome rearrangements in maize induced by alternative transposition of reversed ac/ds termini

Alternative transposition can induce genome rearrangements, including deletions, inverted duplications, inversions, and translocations. To investigate the types and frequency of the rearrangements elicited by a pair of reversed Ac/Ds termini, we isolated and analyzed 100 new mutant alleles derived from two parental alleles that both contain an intact Ac and a fractured Ac (fAc) structure at the maize p1 locus. Mutants were characterized by PCR and sequencing; the results show that nearly 90% (89/100) of the mutant alleles represent structural rearrangements including deletions, inversions, translocations, or rearrangement of the intertransposon sequence (ITS). Among 37 deletions obtained, 20 extend into the external flanking sequences, while 17 delete portions of the intertransposon sequence. Interestingly, one deletion allele that contains only a single nucleotide between the retained Ac and fAc termini is not competent for further alternative transposition events. We propose a new model for the formation of intertransposon deletions through insertion of reversed transposon termini into sister-chromatid sequences. These results document the types and frequencies of genome rearrangements induced by alternative transposition of reversed Ac/Ds termini in maize.     

MAP1B and clathrin are novel interacting partners of the giant cyto-linker dystonin

Dystonin is a large multidomain cytoskeletal-associated protein that plays an essential role in the nervous system. Loss of dystonin results in neuromuscular dysfunction and early death in a mouse mutant called dystonia musculorum. Conserved among related proteins, the plakin domain is a defining feature of all major dystonin isoforms, yet its interactions have not been explored in detail. The purpose of the present study was to identify novel interacting partners of the plakin domain of the neuronal isoform of dystonin (dystonin-a). Newly identified interacting proteins discovered through a pull-down assay were validated using coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays. Microtubule associated protein 1B (MAP1B), a microtubule stabilizing protein, and clathrin heavy chain, the major component of the clathrin triskelion, were identified as interaction partners for dystonin-a. Increased levels of phosphorylated MAP1B suggest a misregulation of MAP1B and a potentially novel component of the dt pathology. This work will further facilitate our understanding of how cytoskeletal proteins can affect and regulate neurodegenerative disorders.