The induction of both long-term potentiation (LTP) and long-term depression (LTD) in the hippocampal CA1 region is triggered by the activation of N-methyl-D-aspartate (NMDA) receptors and the subsequent postsynaptic intracellular Ca2+ increase. However, how NMDA receptor activation differs between LTP and LTD induction is unclear. In the present study, we examined the effects of the magnitude and duration of NMDA receptor activation on the induction of LTP and LTD. Partial blockage of NMDA receptors by a low concentration of aminophosphonovaleric acid (APV) (2 microM) prevented the induction of LTP, but not LTD. In contrast, a high concentration of APV (25 microM) blocked both LTP and LTD. Tetanus stimulation-induced LTP was impaired when hippocampal slices were given the tetanus stimulation for more than 5 min. Under partial blockage of NMDA receptors, the prolonged-tetanus stimulation induced LTD but not LTP. This phenomenon was mimicked by the application of glutamate to the slices. Finally, LTD induced by prolonged activation of NMDA receptors was not affected by inhibition of the desensitization of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. These results suggest that critical differences exist between the induction of LTP and that of LTD in terms of both the magnitude and the duration of NMDA receptor activation. The duration of the increase in intracellular Ca2+ concentration may be critical for determining whether LTP or LTD induction occurs.

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

Alterations in the morphology and movement of mitochondria influence neuronal viability. However, the precise mechanisms of such alterations are unclear. In this study, we showed calcineurin was involved in the regulation of mitochondrial dynamics. Glutamate stimulation inhibited mitochondrial movement and decreased mitochondrial length in neurons. FK506 and cyclosporine A, calcineurin inhibitors, attenuated the effects of glutamate on mitochondrial dynamics. It was also found that glutamate treatment dephosphorylated, a proapoptotic protein, Bad and promoted its translocation to mitochondria in neurons via calcineurin. These results provide important new insights into intracellular signaling pathways that regulate mitochondrial dynamics and neuronal cell death.

PDX-1 plays a central role in differentiation of insulin-producing cells. We previously reported that exogenous PDX-1 protein can permeate cells and induce insulin gene expression in progenitor cells. These data suggest a strategy for facilitating differentiation into insulin-producing cells. Here we show the mechanism of PDX-1 protein transduction. Initially, a punctate cytoplasmic distribution of PDX-1 protein transduction domain (PTD), which co-localized with an endosomal marker, was observed in treated cells. However, homogeneous distribution of PDX-1-PTD was observed in some cells, indicating that PDX-1 is transduced by endocytosis and then released. The experiments using inhibitors suggested that the PDX-1 is transported through the Golgi complex and to the endoplasmic reticulum. Moreover, we observed in real-time PDX-1-PTD release from endosomes. These data suggest that mechanism of transduction of PDX-1 protein is by endocytosis and subsequent release from the endosome homogeneously in cytoplasm and nuclei, and that PDX-1 protein transduction could be a valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells.

The synaptic vesicles keep recycling by the processes of endocytosis and exocytosis to maintain the normal synaptic transmission. The synaptic vesicles are classified as the readily releasable pool (RRP) and the reserve pool (RP). In the endocytosis process, calcineurin (CaN), a Ca2+/calmodulin-dependent protein phosphatase, has been shown to play important roles. However, it is unclear about its roles in different vesicle pools. Here, we investigated the role of CaN in the regulation of vesicle recycling in the RRP and RP. Vesicle recycling was monitored by using fluorescent dyes FM1-43 and FM4-64 in the primary cultures of hippocampal neurons. Inhibition of CaN by FK506 and cyclosporin A suppressed the endocytosis in the RP, but not in the RRP. Inhibition of CaN also restrained the exocytic process triggered by 10 Hz stimulation, but had no effect on 3–5 Hz stimulation-induced exocytosis. FK506 also reduced the total vesicle pool size in the synaptic terminals. A synthesized CaN inhibitory peptide showed the similar effects as FK506 and cyclosporin A. These results revealed a novel mechanism that CaN plays critical roles in the distinct vesicle recycling processes.

Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH2-terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21(WAF1) transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy.

Protein transduction therapy using poly-arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half-life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C-terminal were substituted by arginines. The mutant proteins were effectively delivered in glioma cells and were resistant to Mdm2-mediated ubiquitination. Moreover, the mutant proteins displayed higher transcription regulatory activity and powerful inhibition of the proliferation of glioma cells. These results suggest that ubiquitination-resistant p53 protein therapy may become a new effective cancer therapy.

Astrocytes exhibit dynamic Ca2+ mobilization, such as Ca2+ wave and Ca2+ oscillation, via an inositol 1,4,5-triphosphate-induced Ca2+ release (IICR)-dependent mechanism. The physiological functions of astrocytic Ca2+ mobilization, however, are poorly understood. To investigate this issue, we created a plasmid encoding an enhanced green fluorescent protein-tagged inositol 1,4,5-triphosphate absorbent protein and expressed it in cultured astrocytes. Expression of this protein inhibited both IICR and the Ca2+ wave in cultured astrocytes. By combining this method to the single cell electroporation technique, we were able to inhibit IICR specifically in astrocytes in an astrocyte-neuron co-culture system. Our approach provides a useful tool for direct examination of the physiological role of astrocytic Ca2+ signaling on neuronal function.

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications 1 . Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K + (K ATP ) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca 2+ channel current and increased Ca 2+ influx across the L-type voltage-dependent Ca 2+ channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca 2+ influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the α 1C subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.

on the depression of NMDAR current, suggesting the lack of involvement of the dynamin/clathrin-mediated NMDAR internalization in this process. I mmuno-blotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.

Although memory deficits have been clearly documented in patients with human immunodeficiency virus type-1 (HIV-1) infection, the physiological basis of this dysfunction is poorly understood. We focused on Tat, a viral protein released from HIV-1-infected cells and investigated its effect on spatial learning in adult mice. An intracerebroventricular injection of Tat leads to attenuation of spatial learning accompanied by suppression of long-term potentiation (LTP), the cellular basis of spatial learning, in hippocampal cornu ammonis 1 pyramidal neurons. Tat facilitates extrasynaptic but not synaptic N-methyl-D-aspartate (NMDA) receptor activity. Taken together, these data provide strong evidence that the Tat pathway underlies the development of memory dysfunction in patients with HIV-1 infection and suggest a causal relationship between Tat, the facilitation of extrasynaptic NMDA receptor activity, inhibition of LTP, and attenuation of spatial learning.

Calcineurin and calpain, a Ca2+/calmodulin-dependent protein phosphatase and a Ca2+-dependent cysteine protease, respectively, mediate neuronal cell death through independent cascades. Here, we report that during neuroexcitotoxicity, calcineurin A (CnA) is directly cleaved by calpain in vitro and in vivo, resulting in the enzyme being converted to an active form. Mass spectrometry identified three cleavage sites in CnA, two of which were constitutively active forms. Overexpression of the cleaved CnA induced caspase activity and neuronal cell death. Calpain inhibitors and membrane-permeable calpastatin peptides not only blocked the cleavage of CnA, but also protected against excitotoxic neuronal cell death in vitro and in vivo. These results indicate that CnA is a crucial target for calpain, and the calpain-mediated activation of CnA triggers excitotoxic neurodegeneration. This study established a molecular link between calpain and calcineurin, thereby demonstrating a new mechanism for proteolytical regulation of calcineurin by calpain in response to certain pathological states.

The protein transduction system has been employed for delivery of bioactive proteins into cells via an endocytotic mechanism. However, trapping of endocytosed proteins in the endosome may significantly attenuate biological actions in cells. The present investigation demonstrated that endosomal release of transduced protein could be artificially accelerated by exposure to fluorescent light. Exposure to light at 480 nm stimulated endosomal release of transduced FITC-11 arginine-protein transduction domain (11R-PTD), TAT-PTD and Antennapedia-PTD. Moreover, FITC-11R-p53 protein was released from endosomes following stimulation with light. These data suggest that photo-acceleration is a more efficient strategy in terms of the protein transduction system.

Although memory deficits have been clearly documented in patients with human immunodeficiency virus type-1 (HIV-1) infection, the physiological basis of this dysfunction is poorly understood. We focused on Tat, a viral protein released from HIV-1-infected cells and investigated its effect on spatial learning in adult mice. An intracerebroventricular injection of Tat leads to attenuation of spatial learning accompanied by suppression of long-term potentiation (LTP), the cellular basis of spatial learning, in hippocampal cornu ammonis 1 pyramidal neurons. Tat facilitates extrasynaptic but not synaptic N-methyl-D-aspartate (NMDA) receptor activity. Taken together, these data provide strong evidence that the Tat pathway underlies the development of memory dysfunction in patients with HIV-1 infection and suggest a causal relationship between Tat, the facilitation of extrasynaptic NMDA receptor activity, inhibition of LTP, and attenuation of spatial learning.