Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo

Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions

Attenuation of Soft-Tissue Sarcomas Resistance to the Cytotoxic Action of TNF-α by Restoring p53 Function

BACKGROUND: Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect. METHODOLOGY/PRINCIPAL FINDINGS: We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53(Wt) cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53(Mut) and TP53(Null) cell lines were resistant. TP53 suppression in TP53(Wt) cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53(Null) cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53(Mut) and TP53(Wt)/MDM2(Ampl). In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53(Mut) cells. In TP53(Wt)/MDM2(Ampl) cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM. CONCLUSION/SIGNIFICANCE: More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria–localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death–inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics

Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo

Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions