Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activitydependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high-and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-␣ (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentrationdependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within ϳ 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing antip75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.Cells have the capability of responding to a multitude of signals that it encounters in its extracellular environment. One such signal with widespread pleiotropic actions is the cytokine tumor necrosis factor-␣ (TNF) 1 (1). TNF has been shown to modulate proliferation, differentiation, and apoptotic or necrotic cell death in a number of different cell types (2-4). These disparate responses to TNF are mediated by TNF binding to specific cell surface receptors. Two distinct TNF receptors, type I (p55TNFR) and type II (p75TNFR) (M r 55,000 -60,000 and 70,000 -80,000 in human cells, respectively), have been identified (5, 6), although it remains unclear which of the many responses reported for TNF can be attributed to a specific receptor subtype (4). Moreover, the precise signal transduction pathways for each of these receptor subtypes have yet to be fully delineated. One action of TNF, the induction of apoptosis, is characterized by a discrete set of cellular events regulated by gene expression (7,8). The physiological events accompanying apoptosis include condensation of the chromatin, degradation of DNA through the activation of endogenous nucleases, and dissolution of the cell into small membrane-bound apoptotic vesicles (9, 10). In vivo, these vesicles are phagocytosed by macrophages or other phagocytic cells. Cell death by apoptosis is essential in many physiological processes, including embryonic development of the nervous system (11), oncogenic pathology (12), and clonal selection of hematopoietic cells (13).Conversely, TNF has also been shown to stimulate cellular proliferation in a variety of systems...

The pleitropic actions of tumour necrosis factor-alpha (TNF) are transmitted by the type I 55 kDa TNF receptor (TNFR1) and type II 75 kDa TNF receptor (TNFR2), but the signalling mechanisms elicited by these two receptors are not fully understood. In the present study, we report for the first time subtype-specific differential kinase activation in cell models that respond to TNF by undergoing apoptotic cell death. KYM-1 human rhabdomyosarcoma cells and HeLa human cervical epithelial cells, engineered to overexpress TNFR2, displayed c-Jun N-terminal kinase (JNK) activation by wild-type TNF, a TNFR1-specific TNF mutant and a TNFR2-specific mutant TNF in combination with an agonistic TNFR2-specific monoclonal antiserum. A combination of the TNFR2-specific mutant and agonistic antiserum elicited maximal endogenous or exogenous TNFR2 responsiveness. Moreover, alternative expression of a TNFR2 deletion mutant lacking its cytoplasmic domain rendered the cells unable to activate JNK activity through this receptor subtype. The profile of JNK activation by TNFR1 was more transient than that of TNFR2, with TNFR2-induced JNK activity also being more sensitive to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. Conversely, only activation of the TNFR1 could stimulate mitogen-activated protein kinase (MAPK) or p38 MAPK activities in a time-dependent manner. The role of TNFR2 activation in enhanced apoptotic cell death was confirmed with agonistic monoclonal antisera in cells expressing high levels of TNFR2. Activation of TNFR2 alone elicited cell death, but full TNF-induced death required stimulation of both receptor types. These findings indicate that efficient activation of TNFR2 by soluble TNFs is achievable with co-stimulation by antisera, and that both receptors differentially modulate extracellular signal-regulated kinases contributing to the cytokine's cytotoxic response.

ObjectiveSulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis.MethodsGene expression, histone acetylation, and signaling of the transcription factors NF-E2–related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels.ResultsSFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB–dependent genes. Compared with cytokines alone, SFN (10 μM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 μmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes.ConclusionSFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.