Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity but the underlying signaling mechanisms remain unknown. Here, we show that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into cholesterol-rich lipid rafts from nonraft regions of neuronal plasma membranes. Translocation of TrkB-FL was blocked by Trk inhibitors, suggesting a role of TrkB tyrosine kinase in the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the central nervous system.

IntroductionMultiple myeloma (MM) almost exclusively develops and expands in the bone marrow (BM) and generates devastating bone destruction by osteoclasts (OCs). The bone destruction causes debilitating clinical symptoms including intractable bone pain, disabling multiple fractures, and hypercalcemia. The severity of bone disease correlates with the tumor burden and is one of the major parameters in the Durie and Salomon clinical staging system. Furthermore, the aggressive features of MM bone lesions have contributed significantly to its poor prognosis despite the recent development of intensive chemotherapeutic regimens. 1,2 Therefore, elucidation of the molecular mechanism of bone destruction and tumor progression is essential for the development of effective therapies to improve survival as well as quality of life of patients with MM.Interaction between receptor activator of nuclear factor-B (RANK) expressed on the surface of cells of osteoclastic lineage and RANK ligand expressed on stromal cells plays a key role in the development and activation of OCs, whereas osteoprotegerin, a decoy receptor for RANK ligand secreted from stromal cells, inhibits RANK ligand-RANK signaling. 3,4 MM cells stimulate osteoclastogenesis by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in the BM. [5][6][7] We and others have demonstrated that osteoclastogenic CC chemokines macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1␤ are secreted from most MM cells and play a critical role in the development of MM bone lesions. [8][9][10][11][12] These chemokines directly act on MM cells in an autocrine/paracrine fashion and enhance MM cell adhesion to stromal cells through activation of integrins including very late antigen 4. The interaction between MM cells and stromal cells induces RANK ligand expression by stromal cells, leading to OC differentiation and activation. 8 Furthermore, OCs enhance MM cell growth and survival through a cell-to-cell contact-dependent mechanism that is partially mediated by OC-derived interleukin 6 (IL-6) and osteopontin. 13,14 These observations suggest that interactions of MM cells and OCs form a vicious cycle leading to extensive bone destruction and MM cell expansion.Along with enhanced bone resorption, mineralization is impaired in MM bone lesions. Radiographic examinations show radiolucent lesions without calcification known as "punched-out" lesions. Analyses of bone turnover in patients with MM by biochemical bone markers also suggested an imbalance of bone turnover with enhanced bone resorption and suppressed bone formation. 15 However, the mechanisms of impaired bone formation in bone lesions of patients with MM remain poorly understood.A canonical Wingless-type (Wnt) signaling pathway has been shown to play an important role in osteoblast differentiation. Wnts are secreted cysteine-rich glycoproteins, known as regulators of the differentiation of hematopoietic and mesenchymal cells as well as embryonic development. [16][17][18] Wnt proteins bind to the Frizzle...

Background: Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75 NTR ) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.

Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lyseninresistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wildtype cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.Sphingolipids are ubiquitous constituents of biomembranes in mammalian cells. The most abundant species of sphingolipid in mammalian cells is sphingomyelin (SM), 1 which amounts to 5-20% of total phospholipids. Sphingolipid biosynthesis is initiated by condensation of L-serine with palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) to generate 3-ketodihydrosphingosine (see Ref.1 for a review of sphingolipid biosynthesis). 3-Ketodihydrosphingosine is converted to dihydrosphingosine, which is N-acylated and then dehydrogenated to form ceramide at the endoplasmic reticulum. After moving to the Golgi apparatus, ceramide is converted to sphingomyelin or glycosphingolipids, and these synthesized complex sphingolipids are translocated to the plasma membrane, where they are highly enriched. SPT is suggested to be a key enzyme for regulation of cellular sphingolipid content (1). Regulation of sphingolipid synthesis at the SPT step appears to be relevant to prevention of a harmful accumulation of metabolic sphingolipid intermediates including sphingoid bases and ceramide, since repression of other anabolic steps in the sphingolipid synthetic pathway may cause the intermediate accumulation. Genetic studies have shown that two different genes, LCB1 and LCB2, are required for expression of SPT activity in the yea...