Barry W. Festoff

Cellular distribution of 16S acetylcholinesterase.

Multiple molecular forms of acetylcholinesterase (AChE; EC 3.1.1.7), in crude extracts of various tissues from the rat, were distinguished by velocity sedimentation analysis on linear sucrose gradients. Skeletal muscle samples containing end‐plate regions showed three different forms of AChE with apparent sedimentation coefficients of 16, 10 and 4s. The 16s form was not detected in non‐innervated regions of skeletal muscle, large intestine smooth muscle, whole brain tissue, red blood cells or plasma. Spinal cord, a predominantly motor cranial nerve and mixed (sensory and motor) peripheral nerves contained 16, 10, 6.5 and 4S AChE. Ventral motor roots, supplying the sciatic nerve, contained these four forms of the enzyme, while corresponding dorsal sensory roots were devoid of the 16S form. The 16s‐AChE confined to ventral roots can be attributed totally to motor neurons and not to Schwann cells composing these roots. Whether the 16s‐AChE presently found in motor nerves has chemical identity with that found at motor end‐plates is the basis of future experiments.

Insulin resistance in amyotrophic lateral sclerosis

Over the last 30 years glucose intolerance has been reported in a significant percentage of patients with amyotrophic lateral sclerosis (ALS). Currently, a controversy exists in determining whether the carbohydrate abnormality is disease-specific or secondary to decreased glucose utilization due to muscle atrophy. A reduction in glucose receptor space had been postulated for a number of neuromuscular diseases including ALS. In order to clarify this issue we have estimated in vivo insulin sensitivity, using the euglycemic insulin clamp technique, in ALS patients and two control groups, matched according to percent ideal weight. The results showed that the glucose infusion rate, an estimate of in vivo insulin sensitivity, ws significantly diminished in ALS patients compared to both normal and disease controls. These results demonstrate that the insulin resistance in this disorder cannot be explained by a decrease in glucose-receptor space and suggest a primary carbohydrate aberration in the disease process itself.

The identification of neurotrophic factor as a transferrin.

Partially purified neurotrophic factor (NTF) from chicken nerves comigrated with transferrin and a component in several preparations known to have neurotrophic effects on cultured skeletal muscle cells. One‐dimensional gel electrophoretograms of proteolytic fragments of NTF and fragments obtained from transferrins purified from chicken eggs, serum and embryos were indistinguishable. These purified transferrins, like NTF, all stimulated the incorporation of [3H]thymidine and supported myotube formation to a similar degree as NTF. These studies suggest that NTF is a transferrin‐like protein and that both transferrins and NTF act by initially promoting myoblast proliferation and subsequently supporting myogenesis in chick muscle cultures.

Neuromuscular junction macromolecules in the pathogenesis of amyotrophic lateral sclerosis

An hypothesis regarding the pathogenesis of amyotrophic lateral sclerosis is presented, which places emphasis on extraneural cells. Classical experimental denervation is compared and contrasted with motor neuron disease, both from information in the literature as well as concepts deriving from the hypothesis. Background information regarding neuromuscular junction-specific (16S) acetylcholinesterase and a basal lamina-enriched surface glycoprotein (fibronectin) are presented, which suggest not only their mutual interaction, but likely parallel regulation on muscle cell surfaces by the motor nerve. Since 16S acetylcholinesterase likely contains basal lamina-type collagen and fibronectin specifically associates with collagen, a model relating activation of latent collagenase enzyme in amyotrophic lateral sclerosis is described. It is suggested that continued degeneration, including transneuronal effects, of the motor system ensues from random, continuous loss of nerve-muscle adherence resulting from collagen resorption at the neuromuscular junction.

Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis

When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased. Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE. Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.

Degradation of muscle basement membrane zone by locally generated plasmin

The turnover of basement membrane macromolecules in injured skeletal muscle has not been studied in contrast to other biologic systems undergoing remodeling. Plasminogen activators and other neutral proteases that are able to degrade these basement membrane macromolecules are secreted by cultured muscle cells. We sought to determine if locally released plasminogen activators could act on basement membrane components. Such degradation might be implicated in the disadhesion of nerve from muscle after motor nerve denervation. To test this hypothesis, we first undertook a study of the sensitivity of muscle extracellular matrix antigens following in vitro exposure to various proteases on frozen muscle sections. Fibronectin was found to be most sensitive, followed by type IV collagen and laminin. Of serine proteases, trypsin was the most active but was not selective, digesting matrix and sarcoplasmic components alike in less than 30 min. Purified urokinase was inactive unless plasminogen (also inactive alone) was previously added to tissue sections, at which time only matrix antigens were digested. Little if any observable degradation of sarcoplasmic proteins took place under these conditions. Using a highly sensitive and selective assay, we found that plasminogen activators were present in muscle tissue and increased 8- to 10-fold after 10 days of denervation. Using an extract of denervated muscle in the presence of plasminogen, we observed degradation of matrix antigens. No degradation was observed with control muscle extract. We next evaluated the degradation of these antigens in denervated muscle during a temporal study. The results, analyzed by quantitative image analysis, indicates that with increasing time after denervation a marked decrease of fibronectin and type IV collagen, followed by laminin occurred but, again, only in the present of plasminogen. These results indicate a selective sensitivity of basement membrane antigens of muscle and a role for plasminogen activators in the degradation of these adhesive basement membranes macromolecules after denervation.

Specificity of chicken and mammalian transferrins in myogenesis

Chicken transferrins isolated from eggs, embryo extract, serum or ischiatic-peroneal nerves are able to stimulate incorporation of [3H]thymidine, and promote myogenesis by primary chicken muscle cells in vitro. Mammalian transferrins (bovine, rat, mouse, horse, rabbit, and human) do not promote [3H]thymidine incorporation or myotube development. Comparison of the peptide fragments obtained after chemical or limited proteolytic cleavage demonstrates that the four chicken transferrins are all indistinguishable, but they differ considerably from the mammalian transferrins. The structural differences between chicken and mammalian transferrins probably account for the inability of mammalian transferrins to act as mitogens for, and to support myogenesis of, primary chicken muscle cells.

Monovalent cation transport in myotonic dystrophy

Myotonic dystrophy is a dominantly-inherited disorder which affects skeletal muscle in combination with several other systems. Because of abnormalities in red blood cells, a universal membrane defect has been proposed as the primary disturbance. Erythrocyte cation pump ratios have also been reported to be abnormal. Hyperinsulinemia and glucose intolerance are present in a large number of patients. Since dramatic effects of insulin on membrane cation transport have been shown in several tissues, notably skeletal muscle, we wished first to confirm reports of altered pump ratio in these patients and then to evaluate the effects of insulin on cation fluxes. However, in our experiments myotonic dystrophy patients had normal pump ratios when compared with disease controls.

Environmental influence on altered receptor function in a genetic disease: insulin and glucose affect insulin receptors in myotonic dystrophy.

Insulin action in vivo and insulin binding to monocytes in vitro were correlated in patients with myotonic dystrophy (MyD) and compared with healthy controls. Confirming our previous studies and those of others, the present results show that the glucose infusion rate (DR), an estimate of in vivo insulin sensitivity, was significantly diminished in MyD. At the same per cent of ideal body weight DR in MyD patients was considerably less than controls suggesting that obesity could not solely account for decreased insulin sensitivity in MyD. The relative capacity (RC), and relative affinity (ED50) of the insulin receptor in monocytes was significantly less in patients. The relative affinity (ED50) was improved by changing environmental insulin levels while receptor numbers (RC) were not. Insulin sensitivity and RC showed a trend toward a positive correlation although this did not reach statistical significance. Our data suggest that the alteration of the insulin receptor in MyD is different from obesity and from other disorders of the motor unit such as amyotrophic lateral sclerosis, where insulin sensitivity and RC are reduced but ED50 is unchanged. Thus, in MyD the receptor may be one of the loci where the resistance occurs.

Zeroing in on amyloid proteins in Alzheimer disease therapy

The authors have provided us with a complete review of the approaches to the amyloid proteins of Alzheimers disease in regards to targets for drug therapy. Sufficient information is now available concerning systemic amyloidogenesis, genes for familial Alzheimers disease and the beta amyloid precursor protein, as well as the posttranslational processing requirements for amyloidogenesis and its prevention. Recent excitement concerning the roles for serine proteases and their inhibitors, both in the production and prevention of amyloidogenesis, make this review and its publication in the Neurobiology of Aging extremely timely.