Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Mechanisms of GOLPH3 Associated with the Progression of Gastric Cancer: A Preliminary Study

Study Design

To investigate the specific mechanisms by which Golgi phosphoprotein 3 (GOLPH3) affects the progression of gastric cancer and to explore its clinical significance.

Methods

Immunohistochemical analysis was used to evaluate the correlations between GOLPH3, phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt), phosphorylated p70S6 (p-p70S6), phosphorylated 4E-BP1 (p-4E-BP1) and the clinicopathological features of gastric cancer. The mRNA expression levels of GOLPH3, mTOR, Akt, p70S6 and 4E-BP1 in gastric cancer, carcinoma-adjacent and paired normal tissue were analyzed using RT-PCR. Western blotting was used to determine the protein expression of GOLPH3, p-mTOR, p-Akt, p-p70S6 and p-4E-BP1 in tissues.

Results

High expression protein levels of GOLPH3, p-AKT, p-mTOR, p70S6, p-4E-BP1 were positively associated with histological grade (p<0.05), depth of invasion (p<0.05), distant metastasis (p<0.05) and lymph node involvement (p<0.05). Compared with carcinoma-adjacent and paired normal tissues, the mRNA expression levels of GOLPH3, AKT, mTOR, p70S6 and 4EBP1 in gastric cancer tissues were significantly higher. The protein expression levels of GOLPH3, p-AKT, p-mTOR, p-p70S6 and p-4E-BP1 in gastric cancer tissues were also significantly higher than in carcinoma-adjacent and paired normal tissues. A strong positive correlation was observed between GOLPH3, p-mTOR, p-p70S6 and p-4EBP1 expression (r = 0.410, 0.303 and 0.276, respectively, p<0.05), but no significant correlation between the expression of GOLPH3 and p-Akt was observed.

Conclusions

The GOPLH3 expression level is highly correlated with Akt/mTOR signaling in human gastric cancer samples. GOLPH3 combined with Akt/mTOR signaling activation may play an important role in the development, differentiation, invasion and metastasis of gastric cancer.     

Autocrine activation of cultured macrophages by brain-derived neurotrophic factor

To elucidate a significance of the expression of brain-derived neurotrophic factor (BDNF) in the activated microglia/macrophages of the injured central nervous system, we examined BDNF actions on or BDNF synthesis by macrophages cultured from the mouse peritoneal cavity. They synthesized BDNF and neurotrophin-3 (NT-3) in addition to expressing high-affinity neurotrophin receptors, full-length TrkB (FL), truncated TrkB (TK(-)), and TrkC, thus suggesting an autocrine influence of BDNF and NT-3. BDNF, but not NT-3, enhanced phagocytic activity and stimulated synthesis/secretion of interleukin-1beta in the same manner as lipopolysaccharide (LPS). Furthermore, there was a significant correlation of the phagocytic activity with the expression of BDNF or TrkB (FL). These results imply that the phagocytic activity of macrophages depends on BDNF synthesis and/or TrkB (FL) expression, suggesting that BDNF participates in the activation processes of macrophages by acting in an autocrine manner.     

Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR.     

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 ras

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors, TrkB and TrkC. In contrast to BDNF, NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLCγ were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons. Special issue dedicated to Dr. Hans Thoenen.     

Neurotrophins protect against cytosine arabinoside-induced apoptosis of immature rat cerebellar neurons

Neurotrophin-induced neuroprotection against apoptosis was investigated using immature cultured cerebellar granule cells (CGC) from newborn rat pups. Apoptotic cell death induced by treatment with cytosine arabinoside (AraC) was confirmed by DNA fragmentation and quantified by cell survival assays. AraC was most effective in inducing apoptosis when added to CGC on the day of culture preparation, while less or no effect was observed when added at 24 or 48h after plating, respectively. Pretreatment of CGC cultures for 24h with brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3), robustly protected against AraC neurotoxicity. K252a, an inhibitor of the tropomyosin-related kinase (Trk) tyrosine kinase receptor family which showed no toxicity by itself, blocked BDNF protection of AraC-induced apoptosis in a concentration-dependent manner. Neither protein kinase C activation nor inhibition mimicked or affected BDNF protection against AraC neurotoxicity. BDNF, but not NT-3, treatment of immature CGC caused a marked, but transient activation of Akt through phosphatidylinositol (PI) 3-kinase. The neuroprotective effects of BDNF were suppressed by pretreatment with LY 294002 (a PI 3-kinase inhibitor). BDNF neuroprotection was also preceded by activation of mitogen activated protein kinase (MAPK) and suppressed by two MAPK/ERK (MEK)-selective inhibitors, PD 98059 and U-0126. Moreover, inhibitors of PI 3-kinase and MEK potentiated AraC-induced neurotoxicity. These results show that neurotrophins protect against AraC-induced apoptosis, at least in part, through TrkB-mediated activation of the PI 3-kinase/Akt and MEK signaling pathways.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Neurotrophin-3 and Brain-Derived Neurotrophic Factor Activate Multiple Signal Transduction Events but Are Not Survival Factors for Hippocampal Pyramidal Neurons

Abstract: Expression of the neurotrophin-3 (NT-3) receptor (TrkC) and the effects of NT-3 on signal transduction were investigated in highly enriched populations of embryonic rat hippocampal pyramidal neurons grown in bilaminar cultures. PCR analysis revealed that the predominant trkC isoform is K1, which lacks an insert in the kinase domain. Polyclonal TrkC-specific antibodies stained >90% of the neurons and revealed a single ～145-kDa protein in immunoblots of extracts from adult hippocampus and pyramidal neuron cultures. Addition of NT-3 (50 mg/ml) to these cultures induced the tyrosine phosphorylation of TrkC but not TrkB, as determined by anti-phosphotyrosine staining of immunoprecipitates; thus, all the effects of NT-3 are mediated through TrkC. NT-3 also increased the tyrosine phosphorylation of 42-, 44-, 49-, 55-, 95-, and 145-kDa proteins; the pattern induced by brain-derived neurotrophic factor (BDNF) was similar but not identical to that induced by NT-3, suggesting that subtle differences may exist in signaling by TrkB and TrkC receptors. Immunoprecipitation of p21^ras from ³²P-prelabeled cells showed that NT-3 increased the level of the GTP-bound form of the protein threefold over the control within 5 min. Mitogen-activated protein (MAP) kinase activity was maximally elevated by NT-3 within 2 min and then returned slowly toward baseline over the next 60 min. Tyrosine phosphorylation of phospholipase C-γ increased rapidly after NT-3, suggesting that this enzyme becomes activated. Consistent with this, the neurotrophin rapidly increased protein kinase C activity as well as intracellular Ca²⁺ levels. The effects of both NT-3 and BDNF on Ca²⁺ levels were attenuated in Ca²⁺-free medium, suggesting that both neurotrophins increase Ca²⁺ flux across the plasma membrane as well as release from internal stores. NT-3 also increased c-Fos expression in >80% of the cells; the effect peaked at 30 minand declined to baseline by 120 min. Despite the activation of ras-MAP kinase and phosphoinositide signaling pathways, neither NT-3 nor BDNF alone or in combination could sustain hippocampal pyramidal neurons deprived of glial support. We conclude that in this system NT-3 and BDNF do not appear to be acting as classical “neurotrophic” factors and that activation of the MAP kinase pathway is insufficient for the promotion of neuronal survival.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

螺旋藻对运动疲劳大鼠海马损伤的保护作用及机制研究

目的：研究BDNF/TrkB神经营养信号在运动疲劳大鼠海马神经元损伤中的作用与螺旋藻改善运动致脑海马损伤的作用及其可能的机制。方法：60只雄性SD大鼠随机分为：正常对照组（NC组）、正常+螺旋藻灌胃组（NS组）、运动模型组（EM组）、运动+螺旋藻灌胃组（ES组）、阳性对照组（PC组）,每组12只。EM组、ES组和PC组采用3周的递增式跑台训练建立运动疲劳模型。NC组不施加任何干预,用作对照。NS组和ES组按每天300 mg/kg体重灌胃螺旋藻,PC组以同等体积的人参提取物（1.92 g/kg）灌胃,连续灌胃3周。实验末,用免疫组化和免疫印迹法检测各组大鼠海马BDNF、TrkB、p-TrkB蛋白表达水平,并用尼氏染色法观察海马CA1区形态结构的变化,同时观察大鼠体重等一般情况。结果：与NC组比较,EM组大鼠的体重降低,海马CA1区神经元细胞形态异常且排列紊乱,部分细胞固缩呈不规则变化,部分神经元消失不见,海马BDNF、TrkB和p-TrkB蛋白表达均明显升高（P<0.01）;与EM组比较,ES组大鼠的体重增加,海马神经元损伤得到明显改善,神经元数目和尼氏小体数量增加,神经元排列渐趋规则,形态较完整,ES组海马BDNF、TrkB和p-TrkB蛋白表达均明显升高（P<0.05或P< 0.01）,且与PC组相比,已无明显差别（P>0.05）。结论：BDNF/TrkB神经营养信号可能参与运动致疲劳大鼠海马神经元损伤的修复过程;螺旋藻补充能改善运动疲劳大鼠海马神经元损伤,其原因可能与其上调BDNF和其受体（TrkB）及其受体磷酸化（p-TrkB）蛋白表达而发挥神经保护作用有关。     

Morphoproteomic Profiling of the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Desmoplastic Small Round Cell Tumor (EWS/WT1), Ewing’s Sarcoma (EWS/FLI1) and Wilms’ Tumor(WT1)

Background

Desmoplastic small round cell tumor (DSRCT) is a rare sarcoma in adolescents and young adults. The hallmark of this disease is a EWS-WT1 translocation resulting from apposition of the Ewing’s sarcoma (EWS) gene with the Wilms’ tumor (WT1) gene. We performed morphoproteomic profiling of DSRCT (EWS-WT1), Ewing’s sarcoma (EWS-FLI1) and Wilms’ tumor (WT1) to better understand the signaling pathways for selecting future targeted therapies.

Methodology

This pilot study assessed patients with DSRCT, Wilms’ tumor and Ewing’s sarcoma. Morphoproteomics and immunohistochemical probes were applied to detect: p-mTOR (Ser2448); p-Akt (Ser473); p-ERK1/2 (Thr202/Tyr204); p-STAT3 (Tyr 705); and cell cycle-related analytes along with their negative controls.

Principal Findings

In DSRCT the PI3K/Akt/mTOR pathway is constitutively activated by p-Akt (Ser 473) expression in the nuclear compartment of the tumor cells and p-mTOR phosphorylated on Ser 2448, suggesting mTORC2 (rictor+mTOR) as the dominant form. Ewing’s sarcoma had upregulated p-Akt and p-mTOR, predominantly mTORC2. In Wilm’s tumor, the mTOR pathway is also activated with most tumor cells moderately expressing p-mTOR (Ser 2448) in plasmalemmal and cytoplasmic compartments. This coincides with the constitutive activation of one of the downstream effectors of the mTORC1 signaling pathway, namely p-p70S6K (Thr 389). There was constitutive activation of the Ras/Raf/ERK pathway p-ERK 1/2 (Thr202/Tyr204) expression in the Wilms tumor and metastatic Ewing’s sarcoma, but not in the DSRCT.

Conclusion

Morphoproteomic tumor analyses revealed constitutive activation of the mTOR pathway as evidenced by: (a) expression of phosphorylated (p)-mTOR, p-p70S6K; (b) mTORC 2 in EWS and DSRCT; (c) ERK signaling was seen in the advanced setting indicating these as resistance pathways to IGF1R related therapies. This is the first morphoproteomic study of such pathways in these rare malignancies and may have potential therapeutic implications. Further study using morphoproteomic assessments of these tumors are warranted.     

Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells

The receptor tyrosine kinase TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), are critically important for growth, survival and activity-dependent synaptic strengthening in the central nervous system. These TrkB-mediated actions occur in a highly cell-type specific manner. Here we report that cerebellar Purkinje cells, which are richly endowed with TrkB receptors, develop a normal morphology in trkB-deficient mice. Thus, in contrast to other types of neurons, Purkinje cells do not need TrkB for dendritic growth and spine formation. Instead, we find a moderate delay in the maturation of GABAergic synapses and, more importantly, an abnormal multiple climbing fiber innervation in Purkinje cells in trkB-deficient mice. Thus, our results demonstrate an involvement of TrkB receptors in synapse elimination and reveal a new role for receptor tyrosine kinases in the brain. Electronic Supplementary Materials Supplementary Materials is available in the online version of this article at     

Locomotor Treadmill Training Promotes Soleus Trophism by Mammalian Target of Rapamycin Pathway in Paraplegic Rats

Assisted-treadmill training, may be helpful in promoting muscle mass preservation after incomplete spinal cord injury (SCI). However, biological mechanism involved in this process is still not fully understood. This study investigated the effects of locomotor treadmill training on muscle trophism mediated by protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) in paraplegic rats. Adult female Wistar rats underwent an incomplete thoracic SCI induced by compression using an aneurysm clip. After 7 days, injured animals started a 3-week locomotor treadmill training with body weight-support and manual step help. Soleus trophism was measured by muscle weight and transverse myofiber cross-sectional area (CSA). An enzyme-linked immunosorbent assay (ELISA) and western blot analysis were used to detect brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), Akt, mTOR and p70S6K in paretic soleus. Trained animals did not show locomotor improved, but present an increase in muscle weight and myofiber CSA. Furthermore, the levels of Akt, p70S6K phosphorylation, mTOR and TrkB receptor were increased by training in soleus. In contrast, muscle BDNF levels were significantly reduced after training. The results suggest locomotor treadmill training partially reverts/prevents soleus muscle hypotrophy in rats with SCI. Furthermore, this study provided the first evidence that morphological muscle changes were caused by Akt/mTOR/p70S6K signaling pathway and TrkB up-regulation, which may increase the sensitivity of muscle, reducing autocrine signaling pathway demand of BDNF for cell growth.