Rolf Libelius

Quantitative electromyography of the external anal sphincter in Parkinson's disease and multiple system atrophy

The distinction of multiple system atrophy (MSA) from Parkinson′s disease (PD) can be difficult, especially early in the disease. In MSA degeneration of sacral anterior horn cells (Onufs nucleus) results in denervation‐reinnervation of anal and urethral sphincter muscles, which can be recognized as neurogenic electromyographic (EMG) changes of motor unit potentials. Sphincter EMG has therefore been recommended as a test for distinguishing MSA from PD. Our results confirm the presence of marked neurogenic EMG changes of the external anal sphincter muscle in patients with probable MSA compared to healthy controls. However, in patients with probable PD, our quantitative EMG data show a scatter from normal to marked neurogenic changes and the degree of EMG abnormality is correlated to the duration of the disease. Thus an abnormal sphincter EMG cannot be taken as a strong indicator of MSA rather than PD in the individual patient, especially in long‐standing cases.

T-Tubule endocytosis in dystrophic chicken muscle and its relation to muscle fiber degeneration

SummaryPectoralis muscles from normal and dystrophic chickens were investigated 2h after an i.v. injection of horseradish peroxidase, by cytochemical and biochemical techniques to demonstrate peroxidase activity.Light microscopic examination of dystrophic museles showed that peroxidase activity could be detected inside a population of fibers, in deliminated bodies often restricted to segments of the muscle fiber. Such bodies containing peroxidase were not observed in normal muscle fibers. Electron microscopy of dystrophic muscle fibers revealed that numerous vesicles containing peroxidase were frequently present in fiber regions with signs of cytoplasmic degradation. These vesicles, which occasionally were found to be coated, were 50–100 nm in size and appeared to be derived from t-tubules. Larger (up to 1.7 μm) inclusions containing peroxidase and delimited by a single membrane were also present at degenerating areas of dystrophic muscle fibers. These bodies seemed to be formed by fusion between several primary t-tubule vesicles and probably also lysosomes. Vacuoles containing the peroxidase were frequently encountered. Biochemical determination of horseradish peroxidase activity, performed after extensive washing of the muscle tissue, showed that dystrophic muscles contained about twice as much peroxidase as normal control muscles.It is suggested that endocytosis from t-tubules is an early and essential pathological phenomenon in dystrophic muscle fibers, which may be related to lysosomal function and muscle fiber degeneration.

Trapezius muscle changes unrelated to static work load, Chemical and morphologic controlled studies of 22 women with and without neck pain

From a cross-sectional study of 82 women who were engaged in assembly work that involved static muscle loading of the shoulder muscles, 11 cases with complaints of neck tension (all except 1 arising at work) and 11 individually matched, exposed control cases without neck pain were studied. In addition, 10 matched, unexposed control cases were studied. Upon histochemical examination and study of the trapezius muscle, morphologic changes of type ragged red fibers were found in 8/11 neck-pain cases, in 7/11 exposed controls, and in 4/10 unexposed controls. The pathologic and clinical importance of rare, ragged red fibers in the trapezius muscle thus seems uncertain.

Nogo (Reticulon 4) expression in innervated and denervated mouse skeletal muscle

The nogo gene encodes at least three different proteins, which share a high C-terminal homology with other members of the Reticulon family. Nogo (Reticulon 4) expression has been studied in innervated and denervated mouse hind-limb and hemidiaphragm muscles. A common Nogo A, B, and C probe hybridized to three transcripts, in accordance with human and rat data. Denervation caused decreased Nogo C and increased Nogo A mRNA expression, while Nogo B was not substantially altered. Western blots and immunohistochemistry confirmed the presence of Nogo A-like and Nogo B-like immunoreactivity in muscle. Nogo A-like immunoreactivity increased after denervation and was also present in intramuscular nerves in both innervated and denervated muscle. Nogo B-like immunoreactivity was observed in connective tissue surrounding muscle fibres and nerves. The different Nogo transcripts are produced by both alternative splicing (A and B) and alternative promoter usage (C); both mechanisms seem to be under neural control in skeletal muscle.

Increased endocytosis with lysosomal activation in skeletal muscle of dystrophic mouse.

Endocytosis in dystrophic muscles was studied by a combination of biochemical, radiochemical, and light and electron microscopic techniques. It was observed that the uptake of horseradish peroxidase (HRP) and 3H-Inulin in vitro was increased in leg skeletal muscles from dystrophic mice compared with littermate controls. Endocytosis of HRP in vivo was also increased in dystrophic muscles. When HRP was administered intravenously, light microscopic examination of the muscles showed that the macromolecular tracer was present not only in the extracellular space but also as intracellular deposits in several dystrophic muscle fibers. Ultrastructural examination of these fibers showed HRP to be present in membrane limited bodies of variable size, some of which likely represented secondary lysosomes, located preferentially close to the A-I junction. HRP was also found inside vacuoles which were sometimes in close vicinity to autophagic vacuoles. Primary uptake vesicles containing HRP appeared to originate from the sarcolemma and the transverse tubules. Biochemical determination of lysosomal enzyme activities revealed elevated levels of both cathepsin D and N-acetylglucosaminidase in dystrophic muscles as compared with controls. The results suggest an increased endocytic activity in dystrophic muscles with distribution of exogenous macromolecular tracers into endocytic vesicles and lysosomal structures. The hypothesis is put forward that endocytic activity constitutes an important mechanism of lysosomal activation in dystrophic muscles.

Binding of3H-labelled cobra neurotoxin to cholinergic receptors in fast and slow mammalian muscles

A cobra neurotoxin (Naja naja siamensis 3) acts by blocking acetylcholine receptors of mouse fast and slow muscles in an irreversible way. 3H-labelling of this neurotoxin does not seriously diminish its blocking potency. Its blocking effect on neuromuscular transmission in an isolated phrenic nerve-hemidiaphragm preparation is dependent upon incubation time and toxin concentration. Binding of the toxin to the extensor digitorum longus muscle of the mouse incubatedin vitro is a function of toxin concentration, incubation time and temperature. The binding is an “adsorption” to receptive sites. Longer incubation at +37°C gives a slow increase in binding which does not occur at +4°C. Decamethonium and carbachol, which are known to desensitize the acetylcholine receptor, reduce toxin binding. After denervation an increase in toxin binding per mg wet weight of muscle is observed with peak values at 6 and 21 days following denervation for the fast extensor digitorum longus muscle and the slow soleus muscle, respectively. The highest extrajunctional receptor density of extensor digitorum longus and soleus muscles after denervation was about 3–5× 102 μm−2 respectively. The junctional receptor density of the extensor muscle was estimated at about 1–2×104 μm−2.

Myofibrillar protein and gene expression in acute quadriplegic myopathy.

The dramatic muscle wasting, preferential loss of myosin and impaired muscle function in intensive care unit (ICU) patients with acute quadriplegic myopathy (AQM) have traditionally been suggested to be the result of proteolysis via specific proteolytic pathways. In this study we aim to investigate the mechanisms underlying the preferential loss of thick vs. thin filament proteins and the reassembly of the sarcomere during the recovery process in muscle samples from ICU patients with AQM. Quantitative and qualitative analyses of myofibrillar protein and mRNA expression were analyzed using SDS-PAGE, confocal microscopy, histochemistry and real-time PCR. The present results demonstrate that the transcriptional regulation of myofibrillar protein synthesis plays an important role in the loss of contractile proteins, as well as the recovery of protein levels during clinical improvement, myosin in particular, presumably in concert with proteolytic pathways, but the mechanisms are specific to the different thick and thin filament proteins studied.

Lysosomes in skeletal muscle following denervation: time course of horseradish peroxidase uptake and increase of lysosomal enzymes

SummaryThe in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.

Evidence for endocytotic uptake of cobra neurotoxin in mouse skeletal muscle

An isolated3H-labelled neurotoxin fromNaja naja siamensis binds irreversibly to the extensor digitorum longus muscle of the mousein vitro. The binding consists of an adsorption to cholinergic receptors and of a slower temperature sensitive binding mechanism. The slow binding is markedly stimulated by the presence of cationic proteins (protamine, histone and polylysine) and is blocked at low temperature (+4°C). Vinblastine and colchicine inhibit the stimulatory effect of protamine on the slow binding. Unlabelled neurotoxin blocks the adsorption binding but fails to affect the slow binding. The results suggest that the slow binding of neurotoxin is not associated with the presence of cholinergic receptors but is the result of endocytotic uptake into the muscle cell. Unless properly recognized this uptake will give a considerable overestimate of the number of cholinergic receptors present in the muscle.

Lysosomal activation in mouse skeletal muscle induced by protamine in vitro

SummaryIncubation of mouse skeletal muscle in a physiological Ringer solution containing protamine (60 μg/ml) at +37° C for 1 h induced ultrastructural changes including proliferation of tubular profiles and vesicles at the I-band level close to the A-I junction, formation of numerous acid phosphatase positive lysosomes in the longitudinal sarcoplasmic reticulum and autophagic vacuolation starting at the level of the A-I junction.Biochemical determination of acid phosphatase in the incubated muscles showed that protamine caused an increase in acid phosphatase activity of about 25 % compared to enzyme activities obtained from muscles incubated without protamine at +37°C or with protamine at +4°C.The morphological findings suggest that the vesicles arising adjacent to the A-I junction originate from transverse tubules. Such vesicles, designated as endocytic, may acquire acid phosphatase activity in the longitudinal SR and be active in an autophagic process resulting in large vacuoles. A causal relationship between endocytosis and lysosomal activation is suggested.