Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.

Exploring the mammalian neuromuscular system by analysis of mutations: spinal muscular atrophy and myotonia.

Any biological structure can be studied using mutations that interfere either with its emergence or its function. We investigate spontaneous and induced mutations in the mouse that affect neuromuscular development and function. The wobbler mouse (phenotype WR, genotype wr/wr) suffers from muscular atrophy because of the degeneration of 20-40% of the motoneurones; it is also unable to produce functional spermatozoa. As a step towards positional cloning of the wr gene, we have mapped the locus to proximal chromosome 11, thus excluding CNTF and its receptor as candidates, and suggesting the closely-linked Rab 1 gene encoding a GTP-binding protein as a possibility. In the case of the adr (arrested development of righting response) mouse, which shows hyperexcitability of mature muscle fibres due to a reduction of the 'dampening' function of chloride conductance at resting potential, we have shown that the defect is in the chloride channel gene adr/Clc-1 on chromosome 6. This allowed us to predict via synteny the chromosomal location of human Thomsen's and Becker's myotonias as close to the TCRB gene on human chromosome 7q. The combination of these approaches with gene-targeting approaches will allow genetic analysis of the establishment and structure of the neuromuscular system.

Tumor necrosis factor alpha induces a metalloprotease-disintegrin, ADAM8 (CD 156): implications for neuron-glia interactions during neurodegeneration.

ADAM proteases, defined by extracellular disintegrin and metalloprotease domains, are involved in protein processing and cell-cell interactions. Using wobbler (WR) mutant mice, we investigated the role of ADAMs in neurodegeneration and reactive glia activation in the CNS. We found that ADAM8 (CD 156), a suspected leukocyte adhesion molecule, is expressed in the CNS and highly induced in affected CNS areas of WR mice, in brainstem and spinal cord. ADAM8 mRNA and protein are found at low levels throughout the normal mouse CNS, in neurons and oligodendrocytes. In the WR CNS regions in which neurodegeneration occurs, ADAM8 is induced in neurons, reactive astrocytes, and activated microglia. Similarly, the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is upregulated and shows the same cellular distribution. In primary astrocytes from wild-type and WR mice, in primary cerebellar neurons, and in mouse motoneuron-like NSC19 cells, ADAM8 expression was induced up to 15-fold by mouse TNF-alpha, in a dose-dependent manner. In both cell types, ADAM8 was also induced by human TNF-alpha, indicating that TNF receptor type I (p55) is involved. Induction of ADAM8 mRNA was suppressed by treatment with an interferon-regulating factor 1 (IRF-1) antisense oligonucleotide. We conclude that IRF-1-mediated induction of ADAM8 by TNF-alpha is a signaling pathway relevant for neurodegenerative disorders with glia activation, proposing a role for ADAM8 in cell adhesion during neurodegeneration.

Evidence for the participation of a host enzyme in the activation of poly (A)-Qbeta RNA as an infectious agent.

It has been reported earlier that phage Qbeta RNA (Gilvarg, C., Bollum, F.J. and Weissmann, C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 428-432) elongated at its 3' terminus with up to 100 or more AMP residues retained its full infectivity for Escherichia coli spheroplasts, and that the resulting progeny did not inherit the poly (A) appendage. We now show that while poly (A)-Qbeta RNA appears to function normally as messenger for the synthesis of virus-specific proteins it has lost its capacity to serve as template for Qbeta replicase. Template function could be restored by phosphorolysis with polynucleotide phosphorylase. Taken in conjunction, these results imply that after poly (A)-Qbeta RNA enters the spheroplast a host enzyme (perhaps polynucleotide phosphorylase) removes part or all of the adenylate residues prior to replication of the RNA.

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice.

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.

Dolichos biflorus agglutinin receptors in mouse muscle. II. Biochemical properties in relation to molecular forms of acetylcholinesterase.

A biochemical analysis has been performed on the relationship between the receptors for Dolichos biflorus agglutinin (DBA) and collagen tailed acetylcholinesterase (16S AChE) in mouse skeletal muscle. The molecular forms of AChE were separated by differential salt extraction and by gradient centrifugation. DBA binding activity was measured using a microtiter plate binding assay and affinity chromatography. The 16S form of AChE was bound to DBA, whereas globular forms of AChE were not. However, only a small proportion of 16S AChE was capable of binding to DBA, and most of the DBA binding capacity in muscle extracts was not associated with the 16S AChE. The possible association with the neuromuscular synapse of DBA binding molecules other than 16S AChE is discussed with respect to our previous histochemical study on DBA binding sites in mouse muscle.

Dolichos biflorus agglutinin receptors in mouse muscle. I. Developmental expression in relation to synaptic acetylcholinesterase and to neuromuscular disease.

The lectin (agglutinin) from Dolichos biflorus (DBA) binds selectively to the neuromuscular junction of different vertebrate species. We have examined the synaptic DBA receptors in skeletal muscle during postnatal development and in neuromuscular diseases of the mouse. No DBA binding was found at neuromuscular junctions of muscles from newborn mice, when acetylcholine receptors and acetylcholinesterases were already concentrated at the endplate. Synaptic accumulation of DBA receptors was evident 4 days after birth, and the staining intensity increased until postnatal day 10 to 12 when it reached the adult level. As shown by ultrastructural histochemistry, accessible DBA binding sites were confined to the crests of junctional folds in motor endplates of 4-day-old mice, whereas adult endplates exhibited DBA binding sites in the entire synaptic cleft including the junctional folds. Two neuromuscular hereditary diseases of the mouse, 'wobbler' (WR) and 'motor endplate disease' (MED) were examined. At the light microscopic level, DBA binding was normal in WR and MED endplates. At the ultrastructural level, MED synapses showed reduced junctional folds but unaffected DBA binding capacity.

Mutants of tobacco mosaic virus with temperature-sensitive coat proteins induce heat shock response in tobacco leaves.

We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.

The genomic basis of K(V)3.4 potassium channel mRNA diversity in mice.

K(V)3.4 belongs to the shaw subfamily of shaker-type potassium channels. It conducts fast inactivating, high threshold currents in the central nervous system and in fast-twitch skeletal muscle fibers. The corresponding mouse gene, Kcnc4, consists of five exons spanning a region of 20 kb. Approximately 700 bp of regulatory sequence were delineated. It is GC-rich and lacks typical TATA and CAAT motifs. Instead, seven Sp-1 and three E-box elements define putative regulatory sequences. The mouse K(V)3.4 mRNA has a size of 3639 bp, 1120 bp of which are 3' untranslated region. A transcript initiated from an alternative 5'-exon was identified by RACE and verified by genomic analysis. This isoform, designated K(V)3.4d, is predominantly expressed in skeletal muscle and probably results from alternative promoter usage. It encodes a channel protein with a novel N-terminal cytoplasmic domain. It lacks the conserved sequence motifs encoding the shaw-type tetramerization domain and the 'ball' peptide, which confers fast inactivation properties. Another splice variant, K(V)3.4c, is derived by exon skipping in the C-terminal region and is expressed at similar levels in brain and muscle. These data demonstrate that differential splicing and alternative transcription start sites are utilised to generate a set of K(V)3.4 variants in skeletal muscle and brain, presumably involved in the regulation of excitability.

Expression of chloride channel 1 mRNA in cultured myogenic cells: a marker of myotube maturation.

The chloride channel CIC-1 is required to maintain a normal excitability of mature muscle fibers; its blockade leads to hyperexcitability, the hallmark of the disease myotonia. In mouse and rat myotubes, representing the embryonic stage of muscle, CIC-1 mRNA is not detectable by Northern blotting. From neonatal to adult, CIC-1 expression increases at least fourfold. Using RT-PCR and hybridization on cultured myotubes we found CIC-1 mRNA at a level of 0.4-1.1% of that in mature mouse muscle, and < or = 0.01% in myoblasts, at stages when desmin mRNA levels are already high. The level of CIC-1 mRNA is thus a sensitive and specific indicator of the maturation of skeletal muscle cells.

Differential synthesis by cultured atrial and ventricular rat cardiac myocytes of myosin light chain isoforms.

Dissociated cells from neonatal rat atria and ventricles were cultured in monolayers for 3 days. Newly synthesized 35S-methionine labeled myosin light chain isoforms ALC-1, ALC-2 (atrial) and VLC-1, VLC-2 (ventricular) were identified on 2D gels, and their pattern of synthesis was compared to that of myocard fragments immediately after explanation. ALCs were synthesized in 5- to 10-fold excess over VLCs by atrial cultures, whereas the converse was true for ventricular cultures, with two exceptions: one third of the LC-1 synthesized by ventricular fragments was ALC-1, and dissociated atrial cells synthesized very little LC-2 of either isoform. The former finding corresponds to the relatively high proportion of ALC-1 in neonatal ventricular tissue. We conclude that the regional programme of LC isoform expression is basically retained after tissue explantation and even after dissociation and culturing of cardiac myocytes.

Steroid RU 486 inducible myogenesis by 10T1/2 fibroblastic mouse cells.

For reconstruction or repair of damaged tissues, an artificially regulated switch from proliferation to differentiation would be of great advantage. To achieve conditional myogenesis, we expressed MyoD in mouse C3H 10T1/2 fibroblastic cells, using a gene regulation system based on the synthetic steroid RU 486. By stable co-transfection of a plasmid construct with the RU 486 dependent activator and an integrating inducible MyoD construct, a cell clone, designated 10T-RM, was obtained in which MyoD expression was stringently controlled by RU 486. 12 h after addition of 10 nM RU 486 to 10T-RM cells, saturation levels of MyoD mRNA were observed and >/=2 days later, mRNA for embryonal myosin heavy chain (MyHC(emb)) was abundant and mononucleated cells fused into myotubes.

Expression of the potassium channel KV3.4 in mouse skeletal muscle parallels fiber type maturation and depends on excitation pattern.

We report the detailed expression pattern of the voltage-dependent potassium channel KV3.4 (rat homologue, Raw3) in mouse skeletal muscle. Using semi-quantitative RT-PCR, we show that its expression is detectable at embryonic day 17 and rises to adult levels within 2 weeks after birth. Expression is fiber type-dependent, with mRNA levels being 5-6-fold lower in the mixed slow/fast soleus muscle than in the fast tibialis anterior and extensor digitorum longus muscles. Fast muscles from myotonic mice exhibit low KV3.4 mRNA levels similar to those of wild-type soleus. In denervated extensor digitorum longus, KV3.4 expression declines to perinatal levels. We conclude that KV3.4 expression in mouse skeletal muscle is regulated by the pattern of excitation.

Conservation of the 3'-untranslated region of the Rab1a gene in amniote vertebrates: exceptional structure in marsupials and possible role for posttranscriptional regulation.

The YPT1/RAB1 protein, a key regulator of the intracellular vesicle transport in eukaryotes, is highly conserved in function and amino acid sequence. Here we report that the most highly conserved nucleotide sequence of the Rab1a gene of amniote vertebrates corresponds to the 3'-untranslated region (3'-UTR) of the mRNA. Sequences of 27 species ranging from mammals to sauropsida are >91% identical in this region. Secondary structure prediction procedures applied to the 3'-UTR sequences between positions 750 and 984 and 1428 (mouse cDNA: Y00094), respectively, of the RAB1a mRNAs revealed families of alternative structures around nucleotide position 800 as recurrent features. The two hairpin loops are also predicted for marsupials, despite of their exceptional extension of the A-rich sequence in between. Yet, sequence conservation is much higher than required to conserve secondary structure. Implications for posttranscriptional regulation and protein binding are discussed.

Deletion in the Z-line region of the titin gene in a baby hamster kidney cell line, BHK-21-Bi.

The gene for titin, a 4MDa myofibrillar protein, was analysed in golden hamster DNAs from different sources, using human cDNA probes and PCR. In the DNA from the BHK-21-Bi subline of baby hamster kidney cells, extended sequences coding for Z-line associated domains were missing, indicating a deletion that renders titin non-functional. These sequences were present in the original BHK-21 line and in hamster DNAs. Our finding shows that, due to the absence of selective pressure on a gene's function, genomic deterioration can occur in a permanent cell line and can lead to a loss of overlapping DNA stretches in both autosomes.

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system.

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.

Subtractive cDNA cloning as a tool to analyse secondary effects of a muscle disease. Characterization of affected genes in the myotonic ADR mouse.

In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.

Development of electrical myotonia in the ADR mouse: role of chloride conductance in myotubes and neonatal animals.

In the ADR mouse, the homozygous condition of the autosomal mutation adr, "arrested development of righting response", leads to the symptoms of myotonia. The adr mutation is caused by an insertion of a retroposon into a gene for a chloride channel (adr = Clc-1) that is expressed in adults, but only at very low levels in neonate rodent muscle. In the present study, we investigated the earliest stages of the ADR myotonia. In muscle from 7-day-old ADR mice that can be recognized by inspection, electrical after-activities are distinct by their low frequency (1-5 Hz) and long duration (several minutes) from those recorded in adult muscle. Similar myotonic symptoms could be evoked in muscle fibres from 7 day wildtype mice after substitution of the external chloride with impermeant anions or by activators of protein kinase C. The genotypes of 3-day-old mice cannot be inferred from inspection and, thus, were identified by Southern blotting with a ClC-1 probe. Although no +/+ animal showed characteristic myotonic series, these were seen both in adr/adr and in most adr/+ animals. Thus, due to the low dosage of chloride channels in 3-day-old mouse muscle, the adr mutation appears to be partially dominant rather than fully recessive, as in adult mice. No indication of electrical myotonia could be demonstrated in cultured myotubes, although their pattern of excitability depended on the presence of external chloride ions. We conclude that the low Cl(-)-conductance of myotubes influences excitability but is not controlled by the adr/Clc-1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutual interference of myotonia and muscular dystrophy in the mouse: a study on ADR-MDX double mutants.

For Duchenne muscular dystrophy (DMD, dystrophin deficiency) and Thomsen/Becker myotonia (muscular chloride channel deficiency) genetically homologous mouse models are available, the dystrophin-deficient MDX mouse and the myotonic ADR mouse. Whereas the latter shows more severe symptoms than human myotonia patients, the MDX mouse, in contrast to DMD patients, is only mildly affected. We have introduced, by appropriate breeding, the defect leading to myotonia (Clc1 null mutation, adr allele) into MDX mice, thus creating ADR-MDX double mutants. The expectation was that, due to mechanical stress during myotonic cramps, the ADR status should symptomatically aggravate the muscle fibre necrosis caused by the dystrophin deficiency. The overall symptoms of the double mutants were dominated by myotonia. Weight reduction and premature death rate were higher in ADR-MDX than in ADR mice. Sarcolemmal ruptures as indicated by influx into muscle fibres of serum globulins and injected Evans blue were found with great inter-individual variation in MDX and in ADR-MDX muscles. Affected fibres were found mainly in large groups in MDX but single or in small clusters in ADR-MDX leg muscles. The symptoms of myotonia (aftercontractions, shift towards oxidative fibres) were less pronounced in ADR-MDX than in ADR muscles. Conversely, numbers of damaged fibres as well as the percentage of central nuclei (an indicator of fibre regeneration) were significantly lower in ADR-MDX than in MDX skeletal muscles. Thus it appears that, at the level of the muscle fibre, myotonia and muscular dystrophy attenuate each other.

Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle.

Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.