Tatsuro Mutoh

Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells)

Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.

NGF prevention of neurotoxicity induced by cisplatin, vincristine and taxol depends on toxicity of each drug and NGF treatment schedule:: In vitro study of adult rat sympathetic ganglion explants

The simultaneous administration of nerve growth factor (NGF) has been found to prevent experimental neuropathies induced by anti-cancer drugs such as cisplatin, vincristine and taxol. However, it is clinically important to know whether NGF is beneficial once the neuropathy is already manifest. We established a bioassay system to examine the preventive effects of NGF in various treatment schedules. NGF significantly prevented the inhibition of neurite outgrowth by vincristine and taxol regardless of treatment schedules. The pre-treatment and co-treatment schedules were effective against cisplatin, but the post-treatment schedule was not. With regard to the neurite and nerve cell population densities, only the cisplatin group treated with NGF showed lower values than the control. These results indicate that NGF-treatment is effective for the toxic sympathetic nerve injury induced by vincristine and taxol regardless of the treatment schedule, but is not protective against cisplatin-induced nerve cell injury.

Different characteristics of human herpesvirus 6 encephalitis between primary infection and viral reactivation

BACKGROUND Pathogenesis of human herpesvirus 6 (HHV-6) encephalitis, in particular difference between HHV-6 encephalitis at the time of primary infection and reactivation remains unclear. OBJECTIVES To elucidate the mechanism of HHV-6 encephalitis at the time of primary infection and reactivation. STUDY DESIGN Twenty-two HHV-6 encephalitis patients at the time of primary infection, 6 febrile convulsion (FC) patients caused by HHV-6 infection, and 14 FC patients without HHV-6 infection (non HHV-6 FC) were enrolled. Additionally, 7 stem cell transplant recipients with HHV-6 encephalitis and eight adult controls were also enrolled in this study. Cerebrospinal fluid (CSF) HHV-6 DNA copy numbers and biomarkers levels were compared. RESULTS Low copy number of CSF HHV-6 DNA was detected in 7 of the 22 patients with HHV-6 encephalitis in primary infection, whereas all seven CSF samples collected from post-transplant HHV-6 encephalitis patients contained high viral DNA copy numbers (P<0.001). CSF concentrations of IL-6 (P=0.032), IL-8 (P=0.014), MMP-9 (P=0.004), and TIMP-1 (P=0.002) were significantly higher in patients with HHV-6 encephalitis in primary infection than non-HHV-6 FC. CSF IL-6 (P=0.008), IL-8 (P=0.015), and IL-10 (P=0.019) concentrations were significantly higher in patients with post-transplant HHV-6 encephalitis than adult controls. CONCLUSION The present study suggests that the characteristics of HHV-6 encephalitis are different between HHV-6 encephalitis at the time of primary infection and reactivation in transplant recipients.

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons.

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain‐derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol‐12‐myristate‐13‐acetate (1 μM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain‐derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF‐induced neurite outgrowth from PC12 cells, NGF‐induced activation of ribosomal protein S6 kinase, and NGF‐induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [3H]‐thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca2+ in the medium. 125I‐NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.

Decreased phosphorylation levels of TrkB neurotrophin receptor in the spinal cords from patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of specific populations of cranial and spinal motor neurons. In this study, we examined the expression of the high affinity functional receptor for BDNF, TrkB, and assessed the functional state of TrkB by examining the level of phosphorylation on tyrosine residues in ALS spinal cords. The data showed that TrkB-immunoprecipitates prepared from cell-free lysates of ALS spinal cords by use of an anti-TrkB antibody contained much more TrkB protein than from controls. These TrkB proteins expressed in ALS spinal cords, however, are much less phosphorylated on tyrosine residues than those of controls. Moreover, RT-PCR analysis of TrkB mRNA in ALS spinal cords demonstrated that the expression of Trk B mRNA is also upregulated in ALS spinal cords compared with those of controls. These data strongly suggest that there exists an abnormality in TrkB-mediated intracellular signaling in ALS spinal cords and shed a light on the possibility of the therapeutic intervention by normalizing this intracellular signaling.

HMG-CoA reductase inhibitor-induced L6 myoblast cell death: involvement of the phosphatidylinositol 3-kinase pathway

Our previous studies have shown that the HMG‐CoA reductase inhibitor (HCRI) causes rhabdomyolysis and electrical myotonia in rabbits and also kills L6 myoblasts in culture. In the present study, we analyzed the intracellular signal transduction pathway of HCRI‐induced cell death using L6 myoblasts as a model system. Here, we report that simvastatin, a lipophilic HCRI, efficiently inhibited isoprenylation of Ras proteins and therefore induced translocation of a significant part of Ras proteins from the membrane fraction into the cytosolic fraction within 10 min. With this translocation, PI 3‐kinase activity of the Ras‐bound form both in total and in the membrane fraction was also decreased profoundly. Furthermore, various PI 3‐kinase inhibitors also caused cell death with morphological changes similar to those caused by simvastatin. These results might represent the molecular events of HCRI‐induced cell death, and suggest the significance of PI 3‐kinase activity of the Ras‐bound form in the maintenance of cell viability.

TrkA pathway activation induced by amyloid-beta (Abeta).

Amyloid-beta (Abeta), a cytotoxic fragment of Amyloid Precursor Protein (APP), has been implicated in the etiopathogenesis of Alzheimers disease (AD). Since several neurotrophins signalling pathways may be activated in response to toxic insults, we investigated whether a similar response is triggered also by Abeta. After Abeta (25-35) peptide administration to cultured rat hippocampal neurons, the nerve growth factor (NGF) and its receptor (TrkA) mRNA expression is up-regulated. Moreover, we observe an increased cellular TrkA expression (4.5 fold) and NGF release in the culture medium (5-fold). Concomitantly, TrkA, Akt and glycogen synthase kinase 3beta (Gsk3beta) phosphorylation significantly increase. Interestingly, when cells were treated with Abeta (25-35) in the presence of blocking antibody against NGF, only a partial TrkA activation (2-fold) was observed. These results have been confirmed by using pathophysiological Abeta (1-42) oligomers. Our data provide the evidence that Abeta induces the TrkA pathway activation directly by itself and indirectly promoting NGF secretion.

Role of tyrosine phosphorylation of phospholipase C γ1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts

Our previous studies have shown that the HMG‐CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca2+ mobilization from the Ca2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) γ1. This tyrosine phosphorylation of PLC‐γ1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin‐induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC‐γ1. Interestingly, pretreatment of the cells with U‐73122, a specific inhibitor of PLC, prevented simvastatin‐induced cell death. Thus, these results strongly suggest that simvastatin‐induced tyrosine phosphorylation of PLC‐γ1 plays, at least in part, an important role for the development of simvastatin‐induced cell death.

Stimulated neuronal expression of brain-derived neurotrophic factor by Neurotropin

Expression of brain-derived neurotrophic factor (BDNF) was stimulated in human neuroblastoma SH-SY5Y cells by a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin), an analgesic widely used in Japan for treatment of disorders associated with chronic pain, with the optimal dosage at 10mNU/mL. This stimulation was accompanied by activations of p42/44 MAP kinase, CREB and c-Fos expression. Inhibitors of MAP kinases or PI 3-kinase prevented the stimulatory action of Neurotropin, indicating that neuronal TrkB/CREB pathway mediates the action. Repetitive oral administration of Neurotropin (200NU/kg/day, 3months) prevented the age-dependent decline in hippocampal BDNF expression in Ts65Dn mice, a model of Downs syndrome. This effect was associated with the improvement of spatial cognition of the mice. These results open an intriguing new strategy in which Neurotropin may prove beneficial treatment for neurodegenerative disorders.