Kyoko Koishi

MyoD protein is differentially accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents

MyoD is a muscle-specific transcription factor involved in commitment of cells to myogenesis. MyoD mRNA levels differ between fast and slow muscles, suggesting that MyoD may regulate aspects of fibre type. Here we show that detectable MyoD protein becomes restricted during development to the nuclei of the fastest classes of fibres in fast muscles. myoDm1 mice, in which the myoD gene has been disrupted, show subtle shifts in fibre type of fast muscles toward a slower character, suggesting that MyoD is involved in the maintenance of the fast IIB/IIX fibre type. In contrast, slow muscle shifts to a faster phenotype in myoDm1. Moreover, MD6.0-lacZ transgenic mice with the myoD promoter driving lacZ, show highest beta-galactosidase activity in the fastest fibres of fast muscles, but also express low levels in slow fibres of slow, but not fast, muscles, suggesting distinct regulation of gene expression in slow fibres of fast and slow muscles.

ANTEROGRADE AXONAL TRANSPORT OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR AND ITS RECEPTORS IN RAT HYPOGLOSSAL NERVE

Glial cell line-derived neurotrophic factor is one of the most potent motoneuron survival factors yet identified. Although retrograde transport of trophic factors to the cell body is thought to be an important process in motoneuron survival, the transport pathways that lead to interaction of glial cell line-derived neurotrophic factor with its receptors is not known. We have used a double ligated hypoglossal nerve preparation to investigate transport of endogenous glial cell line-derived neurotrophic factor and its receptors, glial cell line-derived neurotrophic factor family receptor alpha1 and receptor re-arranged during transfection. Glial cell line-derived neurotrophic factor was found to accumulate at the distal ligature, indicating retrograde transport and consistent with its motoneuron survival effects. In addition, we observed accumulation of glial cell line-derived neurotrophic factor and its receptors at the proximal ligature, indicating anterograde transport. This finding is not predicted by neurotrophic theory. Staining for glial cell line-derived neurotrophic factor in the motor axons was punctate, suggesting involvement of transport vesicles. Results obtained using immunohistochemistry and reverse transcription-polymerase chain reaction provide evidence for the synthesis of glial cell line-derived neurotrophic factor and glial cell line-derived neurotrophic factor family receptor alpha1 in Schwann cells and glial cell line-derived neurotrophic factor family receptor alpha1 and receptor re-arranged during transfection in motoneuron cell bodies. When the motor axons were separated from the cell body by avulsion, glial cell line-derived neurotrophic factor remained in the vicinity of the Schwann cells and did not accumulate at the proximal ligature. Our results indicate anterograde transport of Schwann cell-derived glial cell line-derived neurotrophic factor, which is dependent on binding to its cell body-derived receptors. These findings suggest a mechanism for collection of glial cell line-derived neurotrophic factor from multiple Schwann cells which surround motor axons. We propose that in addition to its role in motoneuron survival, glial cell line-derived neurotrophic factor may also modulate local neuronal effects in distal regions of the nerve.

Transforming growth factor-β2 is elevated in skeletal muscle disorders

The transforming growth factor betas (TGF‐βs) are multifunctional growth factors that act on both fibroblasts and myosatellite cells. In rodent models of muscle diseases, high levels of TGF‐β2 are expressed by myogenic cells. We have examined whether the expression of TGF‐β2 is also elevated in diseased human muscles. The disorders examined were Duchenne muscular dystrophy, myotonic dystrophy, myotubular myopathy, spinal muscular atrophy, and amyotrophic lateral sclerosis. The levels of TGF‐β2 immunoreactivity were elevated in atrophic, necrotic, and regenerating fibers and in fibers with central nuclei or cytoplasmic masses, irrespective of whether fibrosis was present. We therefore suggest that TGF‐β2 is important for muscle repair and that the presence of a TGF‐β within a muscle only leads to fibrosis if certain other factors are present.

Müllerian inhibiting substance contributes to sex-linked biases in the brain and behavior

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Müllerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis−/− and Misrii−/− mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.

Transforming Growth Factor-Beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury

The survival of motoneurons is dependent on them receiving continual trophic support from muscle fibres and various other cell types. Numerous putative survival factors have been identified and a set of criteria established by which these candidates can be assessed. These criteria include the need for the factor and its receptors to be in appropriate locations and for the factor or its second message to be retrogradely transported. In this paper, we demonstrate that a multifunctional cytokine, transforming growth factor-beta 2, appears to meet these criteria. The locations of the transforming growth factor-beta 2 and its receptors in the neuromuscular system were determined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Motoneurons were shown to synthesize the three proteins involved in transforming growth factor-beta 2 signalling (types I and II transforming growth factor-beta receptor and betaglycan) and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal. Transforming growth factor-beta 2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2. Double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons. The anterograde transport of both transforming growth factor-beta 2 and its receptors, coupled with the fact that most of a motoneurons mitochondria are located in the axon, raises the issue of whether the repression of the initiation of apoptosis is restricted to the cell body or occurs along the entire length of a neuron.

Transforming growth factor-beta-2 (TGF-β2) is associated with mature rat neuromuscular junctions

The localisation of transforming growth factor-beta-2 (TGF-beta 2) in skeletal muscles was investigated with immunohistochemistry. In neonates, TGF-beta 2 was distributed throughout the muscle fibres but as the fibres matured TGF-beta 2 became restricted to the circumference of a small subpopulation of nuclei. These nuclei were judged to be the subsynaptic nuclei as they lay beneath the plasmalemma and were associated with alpha-bungarotoxin-labelled neuromuscular junctions. These observations point to TGF-beta 2 being either a trophic factor for mature motoneurones or an autocrine regulator of synaptic protein production.

Transforming growth factor beta2 haploinsufficient mice develop age-related nigrostriatal dopamine deficits.

The transforming growth factor-betas (TGF-betas) regulate the induction of dopaminergic neurons and are elevated in the CSF of Parkinsons patients. We report here that mice with TGF-beta2 haploinsufficiency (TGF-beta2+/-) have subclinical defects in the dopaminergic neurons of their substantia nigra pars compacta. At 6 weeks of age, the TGF-beta2+/- mice had 12% fewer dopaminergic neurons than wild-type littermates. No additional loss of neurons occurred during the next 5 months, although striatal dopamine declined to 70% of normal. The level of 3,4-dihydroxphenylacetic acid was normal in the TGF-beta2+/- mice, indicating that a compensatory mechanism maintains dopamine stimulation of their striatum. The TGF-beta2+/- mice had normal sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite having reduced levels of monoamine oxidase-B. These results raise the possibility that people with naturally low levels of TGF-beta2 may have less functional reserve in their nigrostriatal pathway, causing them to be at increased risk of developing Parkinson disease.

TGF-! 2 Attenuates the Injury-Induced Death of Mature Motoneurons

The distributions of transforming growth factor‐betas (TGF‐βs) and their receptors suggest that the TGF‐βs regulate motoneuron survival. This hypothesis was tested by avulsing the hypoglossal nerve of adult rats and perfusing either TGF‐β2 or vehicle adjacent to the hypoglossal nucleus. By 4 weeks, half of the avulsed motoneurons had died. Infusion of 6 ng of TGF‐β2 adjacent to the avulsed motor nucleus caused a significant attenuation of this death. This dose of TGF‐β2 is low compared to that used with GDNF or BDNF in previous studies of avulsed motoneurons, indicating that TGF‐β2 may be one of the more potent survival factors for adult motoneurons. TGF‐β2 was, however, unable to prevent or reduce the axotomy‐induced down regulation of choline acetyltransferase. Other motoneuron survival factors also have a narrow‐spectrum of actions, suggesting that the homeostasis of motoneurons is regulated by a cocktail of growth factors with distinct but partially overlapping actions. J. Neurosci. Res. 62:809–813, 2000.

The non-synaptic expression of transforming growth factor-beta 2 is neurally regulated and varies between skeletal muscle fibre types.

In adult skeletal muscles, transforming growth factor-beta 2 is restricted to the postsynaptic domain of the neuromuscular junction. The various putative functions of this transforming growth factor-beta 2 predict different patterns of transforming growth factor-beta 2 expression in denervated muscles. We therefore denervated rat tibialis anterior, extensor digitorum longus and soleus muscles and examined the expression of transforming growth factor-beta 2 using semi-quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Denervation up-regulated transforming growth factor-beta 2 expression extrasynaptically with little or no effect on synaptic expression. The up-regulation was detectable by one day, had become significant by three days and remained elevated for at least two weeks. This proves that the transforming growth factor-beta 2 associated with the neuromuscular junction is not under neural control and is consistent with transforming growth factor-beta 2 being a trophic factor for motoneurons. This pattern of transforming growth factor-beta 2 expression is similar to that described for other proteins associated with the neuromuscular junction, notably the acetylcholine receptor subunit genes. However, in contrast to the acetylcholine receptor subunit genes, the extent of up-regulation of transforming growth factor-beta 2 varied between fibre types, with the glycolytic IIB fibres being less affected than other fibre types.

BMP6 is axonally transported by motoneurons and supports their survival in vitro.

The regulation of motoneuron survival is only partially elucidated. We have sought new survival factors for motoneuron by analyzing which receptors they produce. We report here that the type II bone morphogenetic receptor (BMPRII) mRNA is one of the most abundant receptor mRNAs in laser microdissected motoneurons. Motoneurons were intensely stained by an anti-BMPRII antibody, indicating the presence of BMPRII protein. One of its ligands (BMP6) supported the survival of motoneurons in vitro. BMP6 was produced by myotubes and mature Schwann cells and was retrogradely transported in mature motor axons. BMP6 thus joins a list of known Schwann-cell-derived regulators of motoneurons, which includes GDNF, CNTF, LIF and TGF-beta2. The control of the production of these factors by Schwann cells and the direction of their movement in motor axons is diverse. This suggests that the multiplicity of motoneuron factors is because cells use different factors to regulate different aspects of motoneuron function.