The Effects Of GM1, IGF-1and Skeletal Muscle Cells On Trk Expression In Drg Neurons

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system and play an important role in signaling events affecting neural development and maturation and are indispensable for the maintenance of brain function and survival. Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Monosialoganglioside (GM1) is a common ganglioside pentasaccharide present on mammalian cell surface. The contents of GM1vary during development of the nervous system. GM1has been considered to have a neurotrophic factor-like activity both in vitro and in vivo. Insulin-like growth factor-1(IGF-1) is a polypeptide growth factor that has a variety of functions in neurons. The capacity of IGF-1on neuronal growth, survival, and differentiation plays an important role in neuronal development and function maintenance. Targets of neuronal innervation play a vital role in regulating the survival and differentiation of the innervating neurons. It has been clarified that the interactions between motor neurons and the target skeletal muscle (SKM) cells. A dorsal root ganglion (DRG) contains cell bodies of primary sensory neurons (PSNs) or primary afferent neurons (PANs) that earn signals from sensory organs toward the center. Muscle spindles convey length information to the central nervous system via DRGs. The three most common types of tyrosine kinase receptor (Trk) receptors (TrkA, TrkB, and TrkC) are expressed in DRG neurons. TrkA, TrkB, and TrkC have high affinity to the binding of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or neurotrophin4/5(NT-4/5). and neurotrophin3(NT-3). respectively. Each of these receptor types is involved in different functional properties of neurons. Whether GM1or IGF-1and target SKM cells affect the expression of distinct Frk receptors is still unclear. The neuromuscular coeultures of DRG neurons and SKM cells were established and used for determining the effects of GM1or IGF-1and target SKM cells on the expression of distinct Trk receptors in DRG neurons. To determine the mechanisms of the effects of GM1or IGF-1on DRG neuronal distinct Trk receptor expression, cell signaling pathways involved in these effects were also assessed by using primary cultured DRG neurons.Part Ⅰ Effects of GM1and skeletal muscle cells on distinct Trk receptor expression in DRG neurons in vitroGMl is a ganglioside which has neurotrophic factor-like biological activities. The target SKM cells play a critical role on neuronal survival and on maintenance of functional properties of neurons. The influence of GMl and SKM on neuronal Trk expression is still unclear. In the present study, embryonic day15(E15) rat DRG neuronal cultures and coeultures of El5rat DRG neurons and newborn rat SKM were established. The following experimental groups were assigned:(1) Neuromuscular coeultures in the absence of GMl:(2) Neuromuscular coeultures in the presence of10μg/ml GMl;(3) DRG neuronal culture in the presence of10μg/ml GMl:(4) DRG neuronal culture in the absence of GMl as control. After6days post-culture, double fluorescent labeling of microtubule-associated protein2(MAP2) and TrkA. TrkB. or TrkC was processed to detection the expression of TrkA. TrkB. or TrkC in DRG neurons. The result showed that GMl promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons: Target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without GMl treatment; GMl and target SKM cells had additional effects on expression of these three Trk receptors. These results imply that GMl and target SKM cells play an important role on distinct Trk receptor expression. The promotion effects of GMl on TrkA and TrkB expression might be by directly activation of TrkA and TrkB. GMl did not modify the percentage of DRG neuron profiles expressing TrkC in dissociate DRG neuronal cultures suggested that GMl failed to displace NT-3binding. TrkC expression was promoted by target SKM cells via the nerve-muscle communication. The results of the present study offered new clues for a better understanding of the association of GMl and target SKM on the expression of Trk receptors.Part Ⅱ Assessment of the signaling pathways of the effects of GM1on Trk receptor expression in DRG neurons in vitroGMl promotes significant functional regulations of the Trk receptor family, which may lead to activation of signal cascades. The present study tried to assess the signaling pathways of the effects of GMl on Trk receptor expression in DRG neurons in vitro. Whether extracellular signal-regulated protein kinase1/2(ERKl/2) and phosphatidylinositol3-kinase (P13K) are involved in the effects of GMl on Trk receptor expression was explored in the present study. El5rat DRG neuronal cultures at2days of culture age were divided into the following5groups.(Ⅰ) GMl group: DRG neurons were treated with10μg/ml GM1for an additional24hours.(2) PD98059+GMl group: DRG neurons were exposed to ERKl/2inhibitor PD98059(10μmol/L)30minutes before GM1(10μg/ml) exposure for an additional24hours.(3) LY294002+GMl group: DRG neurons were exposed to P13K inhibitor LY294002(10μmol/L)30minutes before GMl (10μg/ml) exposure for an additional24hours.(4) PD98059-LY294002+GMl group: DRG neurons were exposed to ERKI/2inhibitor PD98059(10μmol/L) and P13K inhibitor LY294002 (10μmol/L)30minutes before GM1(10μg/ml) exposure for an additional24hours.(5) Control group: DRG neurons were continuously exposed to culture media as a control. After that, the mRNA levels of TrkA, TrkB. and TrkC were determined by real-time PCR analysis. The protein levels of TrkA, TrkB, and TrkC were analyzed with Western blot assay. Double fluorescent labeling of MAP2and TrkA. TrkB. or TrkC was processed to detect the expression of TrkA. TrkB. or TrkC in situ in DRG neurons. The result showed that GM1promoted neurite outgrowth of primary cultured DRG neurons. GM1promoted TrkA and TrkB, but not TrkC protein and mRNA expression of DRG neurons. These effects of GM1were partially blocked by the presence of ERK1/2inhibitor PD98059and/or PI3K inhibitor LY294002. These data implied that the effects of GM1on the expression of TrkA and TrkB and neurite outgrowth of DRG neurons might be via activation of ERK1/2and/or P13K signaling pathways. The new experimental evidence was provided for further study the interactions between GN41and Trk receptors.Part Ⅲ Effects of IGF-1and skeletal muscle cells on distinct Trk receptor expression in DRG neurons in vitroIGF-1is a polypeptide growth factor that has potent neurotrophic and neuroprotective properties. Whether IGF-1affects Trk receptor expression or IGF-1combination with SKM cells affect Trk receptor expression is still unclear. In the present study. H15rat DRG neuronal cultures and cocultures of K15rat DRG neurons and newborn rat SKM were established. The following experimental groups were assigned:(1) Neuromuscular cocultures in the absence of IGF-1;(2) Neuromuscular cocultures in the presence of20nmol/L IGF-1:(3) DRG neuronal culture in the presence of20nmol/L IGF-1:(4) DRG neuronal culture in the absence of IGF-1as control. After6days post-culture, double fluorescent labeling of MAP2and TrkA. TrkB. or TrkC was processed to detection the expression of TrkA. TrkB. or TrkC in DRG neurons. The result showed that IGF"-1promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons. Target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without IGF-1treatment. IGF-1and target SKM cells had additional effects on expression of these three Trk receptors. These results imply that the interactions exist between IGF-1and target SKM cells on distinct Trk receptor expression in DRG neurons. The distinct cellular and molecular mechanisms should be further studied. The results of the present study offered new evidence for further study of the association of IGF-1and target SKM on the expression of Trk receptors.Part Ⅳ Determination of the signaling pathways of the effects of IGF-1on Trk receptor expression in DRG neurons in vitroIGF-1is a polypcptide growth factor that has a variety of functions in neurons. IGF-1has potent neurotrophic and neuroprotective properties. ERKl/2and P13K could be activated by IGF-1. Whether these signaling pathways involved in the effects of IGF-1on Trk receptor expression in DRG neurons remains unknown. In the present study, the signaling pathways of ERKl/2and/or PI3K involved in the effects of IGF-1on Trk receptor expression in primary cultured DRG neurons were determined. According to the experimental design. El5rat DRG neuronal cultures at2days of culture age were divided into the following5groups.(1) IGF-1group: DRG neurons were treated with20nmol/L IGF-1for an additional24hours.(2) PD98059+IGF-1group: DRG neurons were exposed to ERKl/2inhibitor PD98059(10μmol/L)30minutes before IGF-1(20μmol/L) treatment for an additional24hours.(3) LY294002+IGF-1group: DRG neurons were exposed to PI3K inhibitor LY294002(10μmol/L)30minutes before IGF-1(20μmol/L) treatment for an additional24hours.(4) PD98059+LY294002+IGF-1group: DRG neurons were exposed to ERKl/2inhibitor PD98059(10μmol/L) and PI3K inhibitor LY294002(10μmol/L)30minutes before IGF-1(20μmol/L) treatment for an additional24hours.(5) Control group: DRG neurons were continuously exposed to culture media as a control. After that, the mRNA levels of TrkA. TrkB. and TrkC were determined by real-time PCR analysis. The protein levels of TrkA. TrkB, and TrkC were analyzed with Western blot assay. Double fluorescent labeling of MAP2and TrkA. TrkB. or TrkC was processed to detection TrkA. TrkB, or TrkC expression in situ in DRG neurons. The result showed that IGF-1promoted neurite outgrowth of DRG neurons. IGF-1elevated TrkA and TrkB, but not TrkC protein and mRNA levels. Interestingly, neither the FRKI/2inhibitor PD98059nor the PI3K inhibitor LY294002alone blocked the effect of IGF-1. but the use of both inhibitors together was effective. These results indicated that the effect of IGF-1might be via coactivation of both ERK1/2and/or PI3K. signaling pathways. IGF-1signaling might be potential target point for modifying the biological effects mediated by distinct Trk receptors.