ObjectiveA decline in mitochondrial function and biogenesis as well as increased reactive oxygen species (ROS) are important determinants of aging. With advancing age, there is a concomitant reduction in circulating levels of insulin-like growth factor-1 (IGF-1) that is closely associated with neuronal aging and neurodegeneration. In this study, we investigated the effect of the decline in IGF-1 signaling with age on astrocyte mitochondrial metabolism and astrocyte function and its association with learning and memory.MethodsLearning and memory was assessed using the radial arm water maze in young and old mice as well as tamoxifen-inducible astrocyte-specific knockout of IGFR (GFAP-CreTAM/igfrf/f). The impact of IGF-1 signaling on mitochondrial function was evaluated using primary astrocyte cultures from igfrf/f mice using AAV-Cre mediated knockdown using Oroboros respirometry and Seahorse assays.ResultsOur results indicate that a reduction in IGF-1 receptor (IGFR) expression with age is associated with decline in hippocampal-dependent learning and increased gliosis. Astrocyte-specific knockout of IGFR also induced impairments in working memory. Using primary astrocyte cultures, we show that reducing IGF-1 signaling via a 30–50% reduction IGFR expression, comparable to the physiological changes in IGF-1 that occur with age, significantly impaired ATP synthesis. IGFR deficient astrocytes also displayed altered mitochondrial structure and function and increased mitochondrial ROS production associated with the induction of an antioxidant response. However, IGFR deficient astrocytes were more sensitive to H2O2-induced cytotoxicity. Moreover, IGFR deficient astrocytes also showed significantly impaired glucose and Aβ uptake, both critical functions of astrocytes in the brain.ConclusionsRegulation of astrocytic mitochondrial function and redox status by IGF-1 is essential to maintain astrocytic function and coordinate hippocampal-dependent spatial learning. Age-related astrocytic dysfunction caused by diminished IGF-1 signaling may contribute to the pathogenesis of Alzheimer's disease and other age-associated cognitive pathologies.

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A prior study outlined a microscopic technique with which one could quantitate the formation of pinocytic vesicles in cultured mouse macrophages (1). Under these conditions it was evident that vesicle formation responded promptly to factors contained in newborn calf serum. I n addition, through the use of metabolic inhibitors, it was possible to examine certain of the determinants involved in pinosome formation.Largely through the efforts of Holter and his colleagues an increasing body of knowledge has accumulated on the mechanism of pinocytosis in the amoeba (2, 3). A portion of these investigations has been concerned with the induction of channel formation and the resulting uptake of both small and large molecules from the external environment (4). I n general, only charged molecules function as effective stimulators of pinocytosis and these are all thought to bind to the surface coat of the amoeba (5).With the previous studies of the Carlsberg group as a guideline and with the knowledge that newborn calf serum contained pinocytosis-stimulating factors, experiments were undertaken to examine those factors which induced the formation of pinocytic vesicles in mammalian cells. This report will deal with various molecular species which appear to be nonspecific inducers of pinocytic vesicles in cultured mouse macrophages. Materials and MethodsThe basic techniques for the preparation of macrophage cultures, phase-contrast microscopy and the enumeration of pinocytic vesicles, have been reported in a previous article (1).The Study of Inducers of Pinocytosis.--Macrophage cultures were incubated in a 500/0 newborn calf serum (NBCS)-No. 199 medium for either 24 or 48 hr at 37°C. At the time of an experiment the medium was removed from Leighton tubes by means of a fine tipped pipette. Residual serum components were then washed from the culture vessel by two lavages with warm, medium 199. After the second washing, 1.0 ml of 1% NBCS-No. 199 was added to the cultures. This was the basic medium to which all inducers were then added. It was the usual practice to wash and add 1% serum medium to all the tubes of a given experiment. This pro-

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

A prior publication described the pinocytosis-inducing properties of a variety of both high and low molecular weight molecules (1). This study indicated that anionic molecules stimulated the formation of pinocytic vesicles whereas neutral and cationic species had in most instances little influence on this parameter of endocytic activity. Some of the most potent inducers proved to be macroanions and these molecules were thought to function by virtue of a physicochemical interaction with a membrane receptor. This view was suggested by the previous studies on the amoeba (2) and the similar molarities at which many of the macroanious stimulated a response in the macrophage system.One of the groups of macroanions studied in these experiments were the nucleic adds. Both ribonucleic and deoxyribonucleic acid enhanced vesicle flow in cultured mouse macrophages. This prompted further studies with both nucleic acid derivatives and high energy phosphate intermediates. This article will document the unique morphological and sfimulatory effects of adenosine and its Y-phosphates on macrophage pinocytic activity. Materials and MethodsThe preparation of macrophage cultures, enumeration of pinocytic vesicles, fixation and photographic procedures have been discussed (1,3). In these experiments cultures were maintained for either 24 or 48 hr in 50% newborn calf serum-No. 199 medium. Samples of newborn calf serum were obtained from either Microbiological

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter Allopregnanolone levels, an important neurosteroid in the brain. We assessed the levels and regulation of allopregnanolone and the effects of allopregnanolone supplementation on cognitive function in 4 month old and 24 month old male C57BL/6 mice. With age, the expression of enzymes involved in the allopregnanolone synthetic pathway were decreased and corticosterone synthesis increased. Supplementation of allopregnanolone improved cognitive function. Interestingly, IL-6 infusion in young animals significantly reduced the production of allopregnanolone compared to controls. Of note, inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein gp130 (sgp130), significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of allopregnanolone and increases corticosterone levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in allopregnanolone levels and an increase in corticosterone. Furthermore, our results indicate that allopregnanolone is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

Aims: Human c-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize c-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX 3 C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell.

Background:The endocytic pathway spatially regulates EGFR signaling. Results: Activated, cell surface, EGFRs stimulate Src and c-Cbl better than intracellular receptors and increase receptor ubiquitylation. Conclusion: Sustained signaling from the plasma membrane enhances receptor ubiquitylation, a key component of EGFR down-regulation. Significance: Cell surface EGFRs signal key regulatory mechanisms for receptor degradation.