James L. McManaman

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: Effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.

Multiple Neurotrophic Factors from Skeletal Muscle: Demonstration of Effects of Basic Fibroblast Growth Factor and Comparisons with the 22-Kilodalton Choline Acetyltransferase Development Factor

Abstract: Extracts of skeletal muscle contain chromato‐graphically distinct molecules that enhance the cholinergic development of cultured embryonic rat spinal cord neurons. We have recently purified a 20–22 kilodalton anionic polypeptide choline acetyltransferase (ChAT) development factor (CDF) from rat skeletal muscle extracts that stimulates the development of ChAT activity in rat spinal cord cultures. The maximum increase in the level of ChAT activity achieved by this factor, however, is less than that achieved by the addition of the crude extract. We now show that muscle extract also contains mitogenic activity that is immunologically related to basic fibroblast growth factor (bFGF) and also that recombinant bFGF stimulates ChAT development in rat spinal cord cultures. bFGF, however, differs from CDF in its physicochemical, chromatographic, and immunological properties and by its action on nonneuronal cells. Individually, CDF and bFGF each enhance the level of ChAT activityin rat spinal cord cultures two‐ to threefold after 2 days of treatment. However, their combined actions result in a five‐to sixfold enhancement of ChAT activity, suggesting that they are affecting cholinergic development through different means. The demonstration that extracts of rat skeletal muscle contain two biochemically and immunologically distinct polypeptides, with additive effects on cultured embryonic spinal cord neurons, provides additional evidence for the involvement of multiple target‐derived neurotrophic factors in the regulation of cholinergic development

Temporal and spatial expression of ciliary neurotrophic factor after peripheral nerve injury.

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions.

The effect of calcium on acetylcholine receptor synthesis.

The level of cytoplasmic calcium has been proposed to act as a regulator of acetylcholine receptor synthesis (Betz, H., and J. P. Changeaux (1979) Nature 278: 749–751). However, there is little known about the effect of altered calcium levels on the metabolism of the acetylcholine receptor. We have investigated the effect of decreased extracellular calcium on the metabolism of acetylcholine receptors in cultured rat myotubes. Our results show that the acetylcholine receptor levels on the surface of myotubes were decreased 25 to 30% following overnight incubation in calcium-deficient medium. In contrast, creatine phosphokinase activity levels and total protein synthesis were unaffected. Calcium depletion did not change the rate of receptor degradation significantly (0.037 hr-1, compared to 0.033 hr-1 for control cells) but dramatically decreased the rate of incorporation of new acetylcholine receptors into the plasma membrane. The time course for incorporation of new acetylcholine receptors into the plasma membrane of calcium-depleted cells was similar to control cells treated with cycloheximide, suggesting that de novo receptor synthesis was inhibited. These results indicate that intracellular calcium levels and acetylcholine receptor synthesis are not related in a simple reciprocal fashion and suggest that the regulation of acetylcholine receptor levels involves more than one intracellular compartment of calcium.

Regulation of Tyrosine Hydroxylase Gene Expression in IMR‐32 Neuroblastoma Cells by Basic Fibroblast Growth Factor and Ciliary Neurotrophic Factor

Abstract: The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR‐32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two‐ to threefold over those found in bFGF‐treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8‐(4‐chlorophenylthio)‐cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.

Inhibitors of membrane depolarization regulate acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism

The inhibition of membrane depolarization by tetrodotoxin or the local anesthetic benzocaine elevates the acetylcholine receptor levels in cultured myotubes. The elevated acetylcholine receptor levels are due to increased receptor synthesis rather than to decreased degradation. The effects of tetrodotoxin and benzocaine on acetylcholine receptor levels are not additive, and are not inhibited by exogenously added cyclic GMP analogues or by elevated intracellular levels of cyclic GMP. However, the stimulation of acetylcholine receptor levels by tetrodotoxin or benzocaine is reversed by the addition of the calcium ionophore A23187. In contrast, tetrodotoxin or benzocaine stimulated acetylcholine receptor synthesis beyond the maximal stimulation produced by cholera toxin. These results suggest that the inhibition of membrane depolarization elevates acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism.

Developmental discord among markers for cholinergic differentiation: In vitro time courses for early expression and responses to skeletal muscle extract

The effects of skeletal muscle extract on the development of CAT, ACh synthesis, high affinity choline uptake, and AChE activities were studied in dissociated ventral spinal cord cultures prepared from 14-day gestational rat embryos. In the absence of muscle extract, the development of CAT and AChE follow biphasic time courses in which they show initial declines followed by periods of steadily increasing activity. In contrast, ACh synthesis and high affinity choline uptake both gradually increase throughout the entire culture period. The presence of muscle extract both prevents the initial decline of CAT and AChE as well as stimulates the rates of development of all four cholinergic markers; however, the degrees and time courses of stimulation differ markedly. The effects of muscle extract on the kinetic and pharmacological properties of ACh synthesis and choline uptake in rat ventral cord cultures were also investigated. Cells treated with muscle extract for 2 days express both high affinity (Km = 1.6 microM) and low affinity (Km = 22 microM) choline uptake mechanisms. Control cells, on the other hand, express only low affinity uptake at this stage but develop a high affinity uptake mechanism by Day 7. During this time both ACh synthesis and high affinity choline uptake become increasingly sensitive to inhibition by hemicholinium-3. These results demonstrate that skeletal muscle factors enhance the development of cholinergic properties in embryonic spinal cord cultures. However, differences in sensitivity to muscle extract concentration, time courses of development, and degrees of stimulation suggest that these changes may involve distinct cellular mechanisms which are differentially affected by skeletal muscle factors.

Low-molecular-weight peptide stimulates cholinergic development in ventral spinal cord cultures.

Skeletal muscle extract contains a previously undocumented 1300- to 1500-Da neurotrophic factor. Incubation of ventral spinal cord neurons in the presence of this factor enhances the rate of de novo acetylcholine synthesis two- to threefold over control cells, after 6 days in culture. This effect on cholinergic activity appears to be selective, since incubation with the factor results in only slight elevations of lactate dehydrogenase activity and DNA content, and no increase in the acetylcholinesterase activity. The 1300- to 1500-Da factor is acid-stable and partially sensitive to proteolysis by proteinase K, Staphylococcus aureus V8 protease, and subtilisin, but insensitive to trypsin. These results indicate that the active moiety is a peptide. The importance of peptides as neurotransmitters or neuromodulators is well accepted, but their role in the regulation of neuronal development is not widely appreciated. The present cholinergic neurotrophic peptide is distinct from previously characterized cholinergic trophic factors and represents the first example of a small, target-derived peptide which influences cholinergic development.

Skeletal muscle proteins stimulate cholinergic differentiation of human neuroblastoma cells.

Abstract: Extracts of rat skeletal muscle contain substances that enhance the development of choline acetyltransferase (ChAT) in the cholinergic human neuroblastoma cell line LA‐N‐2. The ChAT enhancing activity in muscle extract was purified to homogeneity by preparative gel electrophoresis and reverse‐phase HPLC. The active factor is biochemically and immunologically identical to ChAT development factor, (CDF), the skeletal muscle factor that enhances ChAT activity in enriched cultures of embryonic rat motoneurons and rescues motoneurons from naturally occurring cell death in vivo. CDF increases the specific ChAT activity of LA‐N‐2 cells fivefold after 6 days in culture, but does not affect their growth or metabolic activity. Basic fibroblast growth factor also increases ChAT activity in LA‐N‐2 cells and its effect is additive with that of CDF. In contrast, neither insulin‐like growth factor‐1, epidermal growth factor, nor nerve growth factor affected the ChAT activity of LA‐N‐2 cells. Our study demonstrates for the first time that CDF can directly affect the development of neuronal properties in a homogeneous population of cells, and that the effects of CDF are separate from those of other types of trophic factors.

Muscle-derived trophic factors influencing cholinergic neurons in vitro and in vivo

Publisher Summary This chapter review the effects of two different factors derived from muscle which enhance cholinergic properties of neurons in vitro. In the absence of muscle extract the development of choline acetyltransferase (CAT) in tissue culture follows a biphasic time course in which there is an initial decrease in CAT activity followed by a period of steadily increasing activity. The addition of muscle extract to the cultures prevents the initial decline and stimulates the subsequent development of CAT activity. A factor (cholinergic development factor, CDF) in the muscle extract which produces such effects has been purified and partially characterized. While CDF was being purified from rat skeletal muscle, attempts were made to purify neurotrophic activity from autopsied human skeletal muscle. Chick ciliary neurons were used to assay the enhancement of acetylcholine synthesis, CAT. Although neurotrophic factors may exhibit a range of specific behaviors in vitro such as neuron survival or enhancement of cholinergic activity, for complete validation such factors must be shown to be capable of influencing neuronal survival in vivo. An in vivo effect could eliminate a trivial explanation of the in vitro phenomena, i.e. that purified extracts may provide essential components missing or perturbed in the tissue culture environment, or that they act in a manner normally inoperative in vivo.