Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

Target determination of neurotransmitter phenotype in sympathetic neurons

While the majority of sympathetic neurons are noradrenergic, a minority population are cholinergic. At least one population of cholinergic sympathetic neurons arises during development by a target-dependent conversion from an initial noradrenergic phenotype. Evidence for retrograde specification has been obtained from transplantation studies in which sympathetic neurons that normally express a noradrenergic phenotype throughout life were induced to innervate sweat glands, a target normally innervated by cholinergic sympathetic neurons. This was accomplished by transplanting footpad skin containing sweat gland primordia from early postnatal donor rats to the hairy skin region of host rats. The sympathetic neurons innervating the novel target decreased their expression of noradrenergif traints and developed choline acetyltransferase (ChAT) activity. In addition, many sweat gland-associated fibers acquired acetylcholinesterase (AChE) staining and VIP immunoreactivity. These studies indicated that sympathetic neurons in vivo alter their neurotransmitter phenotype in response to novel envronmental signals and that sweat glands play a critical role in the cholinergic and peptidergic differentiation of the sympathetic neurons that innervate them. The sweat gland-derived cholinergic differentiation factor is distinct from leukemia inhibitory factor and ciliary neurotrophic factor, two well-characterized cytokines that alter the neurotransmitter properties of cultured sympathetic neurons in a similar fashion. Recent studies indicate that anterograde signalling is also important for the establishment of functional synapses in this system. We have found that the production of cholinergic differentiation activity by sweat glands required sympathetic innervation, and the acquisition and maintenance of secretory competence by sweat glands depends upon functional cholinergic innervation. 1994 John Wiley & Sons, Inc.     

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: The role of cholinergic differentiation factor/leukemia inhibitory factor

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.     

GM2 Promotes Ciliary Neurotrophic Factor-Dependent Rescue of Immortalized Motor Neuron-Like Cell (NSC-34)

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblasoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.     

睫状神经营养因子对大鼠去神经骨骼肌的营养作用

目的：了解睫状神经营养因子（CNTF）对去神经引起的肌肉萎缩的治疗作用。方法：离断SD大鼠一侧坐骨神经,连续给予CNTF20d,观察肌肉湿重、蛋白含量、肌纤维横截面积、收缩性能和残肢程度。结果：①给予0.2mg/kg的CNTF,可使损务侧肌纤维横截面积增加35％,肌肉湿重增加38％,胫前肌总蛋白含量增加24％,腓长肌强直收缩强度提高40％,显著改善肢残程度;②0.2mg/kg的CNTF作用明显强于0.05mg/kg的CNTF;③此目鱼肌（慢肌）比伸趾长肌（快肌）对CNTF更敏感。结论：CNTF能显著改善成年大鼠坐骨神经离断后骨骼肌的萎缩和功能丧失,该效应的强弱与用药剂量和肌肉类型有关。     

Ciliary neurotrophic factor promotes inactivation of muscle Ca2+ channels via PKC

The actions of the ciliary neurotrophic factor (CNTF) were assessed on adult mouse skeletal muscle L-type Ca2+ currents and on Ca2+ release from sarcoplasmic reticulum. Currents were measured with the whole cell patch clamp technique. Ca2+ signals in response to single action potentials were recorded with Fluo3-AM. CNTF (20 ng/ml) reversibly reduced the amplitude of Ca2+ channel currents by 50% within 15 min. In addition, CNTF greatly increased the rate of inactivation during depolarizing pulses and shifted the steady state inactivation curve by -12 mV. The effects of CNTF were mimicked by the PKC activator PMA and prevented by the PKC-inhibitor chelerythrine. In contrast to the effects on the Ca2+ conductance, charge movement and Ca2+ signals remained unaffected by CNTF. These results suggest that CNTF can rapidly decrease muscle Ca2+ channel currents by promoting inactivation, probably through an intracellular PKC-dependent mechanism.     

Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells

The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons.     

A putative physiological role of hypothalamic CNTF in the control of energy homeostasis

Administration of CNTF durably reduces food intake and body weight in obese humans and rodent models. However, the involvement of endogenous CNTF in the central regulation of energy homeostasis needs to be elucidated. Here, we demonstrate that CNTF and its receptor are expressed in the arcuate nucleus, a key hypothalamic region controlling food intake, and that CNTF levels are inversely correlated to body weight in rats fed a high-sucrose diet. Thus endogenous CNTF may act, in some individuals, as a protective factor against weight gain during hypercaloric diet and could account for individual differences in the susceptibility to obesity.     

Alterations in Gene Expression Associated with Primary Demyelination and Remyelination in the Peripheral Nervous System

Primary demyelination is an important component of a number of human diseases and toxic neuropathies. Animal models of primary demyelination are useful for isolating processes involved in myelin breakdown and remyelination because the complicating events associated with axonal degeneration and regeneration are not present. The tellurium neuropathy model has proven especially useful in this respect. Tellurium specifically blocks synthesis of cholesterol, a major component of PNS myelin. The resulting cholesterol deficit in myelin-producing Schwann cells rapidly leads to synchronous primary demyelination of the sciatic nerve, which is followed by rapid synchronous remyelination when tellurium exposure is discontinued. Known alterations in gene expression for myelin proteins and for other proteins involved in the sequence of events associated with demyelination and subsequent remyelination in the PNS are reviewed, and new data regarding gene expression changes during tellurium neuropathy are presented and discussed.