A critical component of nervous system development is synapse elimination during early postnatal life, a process known to depend on neuronal activity. Changes in synaptic strength in the form of long-term potentiation (LTP) and long-term depression (LTD) correlate with dendritic spine enlargement or shrinkage, respectively, but whether LTD can lead to an actual separation of the synaptic structures when the spine shrinks or is lost remains unknown. Here, we addressed this issue by using concurrent imaging and electrophysiological recording of live synapses. Slices of rat hippocampus were cultured on multielectrode arrays, and the neurons were labeled with genes encoding red or green fluorescent proteins to visualize presynaptic and postsynaptic neuronal processes, respectively. LTD-inducing stimulation led to a reduction in the synaptic green and red colocalization, and, in many cases, it induced a complete separation of the presynaptic bouton from the dendritic spine. This type of synapse loss was associated with smaller initial spine size and greater synaptic depression but not spine shrinkage during LTD. All cases of synapse separation were observed without an accompanying loss of the spine during this period. We suggest that repeated low-frequency stimulation simultaneous with LTD induction is capable of restructuring synaptic contacts. Future work with this model will be able to provide critical insight into the molecular mechanisms of activityand experience-dependent refinement of brain circuitry during development.verproduction of synapses and their subsequent elimination through activity-and experience-dependent processes are critical for refinement of neuronal circuits during development (1). One useful model of synapse elimination has been the mammalian visual cortex, where pruning of geniculocortical synapses takes place in response to monocular visual deprivation (MD) during postnatal critical periods (2). Neuronal activity is essential for this type of synapse elimination because blockade of action potentials in the cortex with tetrodotoxin (TTX) completely prevents synaptic loss and restructuring after MD (3, 4). Moreover, the loss of synapses in several models requires NMDA receptors, suggesting that postsynaptic processes also may be important (5-7). These findings indicate that synapse elimination is an active process, not merely a function of nonuse (8).Structural plasticity such as that described after MD has been associated with retraction of entire axonal branches (9, 10), but it is likely this process begins at the synaptic level. How might synapses be eliminated in such an activity-dependent manner? As predicted by the Beinenstock, Cooper, and Munroe (BCM) theory (11, 12), low-frequency synaptic stimulation weakens glutamatergic synapses in hippocampal slices [long-term depression (LTD)] (13) and such synaptic depression might provide the first step in the experience-dependent refinement of neural circuits (11, 13). Current biochemical and physiological evidence supports a link between the...

Purpose Mitochondrial glycerophosphate dehydrogenase (mGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin (MF) in the liver. There are no data on the expression and role of mGPDH as a metformin target in cancer. In this study, we evaluated mGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism. Experimental design We analyzed mGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer-derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on mGPDH expression were determined by qRT-PCR and western blot. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and mGPDH expression in metastatic thyroid cancer mouse models. Results We show for the first time that mGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that mGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of mGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high mGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth inhibitory effects of metformin in vitro and in vivo. Conclusion Our study established mGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin.

BCL2 is best known as a multifunctional anti-apoptotic protein. However, little is known about its role in cell adhesive and motility events. Here, we show that BCL2 may play a role in the regulation of cell adhesion, spreading, and motility. When BCL2 was overexpressed in cultured murine and human cell lines, cell spreading, adhesion, and motility were impaired. Consistent with these results, loss of Bcl2 resulted in higher motility observed in Bcl2 null mouse embryonic fibroblast cells compared to its wild type. The mechanism of BCL2 regulation of cell adhesion and motility may involve formation of a complex containing BCL2, actin and gelsolin, which appears to functioally decrease gelsolin-severing activity. We have observed that the lysate from MCF-7 cells and NIH3T3 cells that overexpressed BCL2 enhanced actin polymerization in cell-free in vitro assays. Confocal immunofluorescent localization of BCL2 and F-actin during spreading consistently showed that increased expression of BCL2 resulted in increased F-actin polymerization. Thus, formation of BCL2 and gelsolin complexes (which possibly contains other proteins) appears to play a critical role in regulation of cell adhesion and migration. Given the established correlation of cell motility with cancer metastasis, this result may explain why expression of BCL2 in some tumor cell types reduces the potential for metastasis and shows improved patients prognosis.

We demonstrate that physiologically relevant perturbations in the osmotic environment rheostatically regulate a gatekeeping function for the nucleolus that controls the spatial dynamics and functions of nucleolin. HeLa cells and U2-OS osteosarcoma cells were osmotically challenged with 100 -200 mM sorbitol, and the intranuclear distribution of nucleolin was monitored by confocal microscopy. Nucleolin that normally resides in the innermost fibrillar core of the nucleolus, where it assists rDNA transcription and replication, was expelled within 30 min of sorbitol addition. The nucleolin was transferred into the nucleoplasm, but it distributed there non-uniformly; locally high levels accumulated in 4,6-diamidino-2-phenylindole-negative zones containing euchromatic (transcriptionally active) DNA. Inositol pyrophosphates also responded within 30 min of hyperosmotic stress: levels of bisdiphosphoinositol tetrakisphosphate increased 6-fold, and this was matched by decreased levels of its precursor, diphosphoinositol pentakisphosphate. Such fluctuations in inositol pyrophosphate levels are of considerable interest, because, according to previously published in vitro data, they regulate the degree of phosphorylation of nucleolin through a novel kinase-independent phosphotransferase reaction (Saiardi, A., Bhandari, A., Resnick, R., Cain, A., Snowman, A. M., and Snyder, S. H. (2004) Science 306, 2101-2105). However, by pharmacologically intervening in inositol pyrophosphate metabolism, we found that it did not supervise the osmotically driven switch in the biological activities of nucleolin in vivo.