To determine whether changes in glycogen synthase kinase-3beta (GSK-3beta) phosphorylation contribute to muscle hypertrophy, we delineated the effects of GSK-3beta activity on C(2)C(12) myotube size. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible gene activity and possible modulation of NFAT activation by GSK-3beta. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., both inhibit GSK-3beta activity) increased the area of C(2)C(12) myotubes by 80 and 85%, respectively. The application of IGF-I (250 ng/ml) elevated GSK-3beta phosphorylation and reduced GSK-3beta kinase activity by approximately 800% and approximately 25%, respectively. LY-294002 (100 microM) and wortmannin (150 microM), specific inhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-induced GSK-3beta phosphorylation by 67 and 92%, respectively. IGF-I suppressed the kinase activity of GSK-3beta. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporter activity. Cotransfection of a constitutively active GSK-3beta (cGSK-3beta) inhibited the induction by IGF-I of NFAT-inducible reporter activity. LiCl, which inhibits GSK-3beta, removed the block by cGSK-3beta on IGF-I-inducible NFAT-responsive reporter gene activity. These data suggest that the IGF-I-induced increase in skeletal myotube size is signaled, in part, through the inhibition of GSK-3beta.

Currently, the repertoire of cellular and molecular pathways that control skeletal muscle atrophy and hypertrophy are not well defined. It is possible that intracellular regulatory signaling pathways are active at different times during the muscle hypertrophy process. The hypothesis of the given experiments was that cellular signals related to protein translation would be active at early time points of skeletal muscle regrowth, whereas transcriptional signals would be active at later time points of skeletal muscle regrowth. The phosphorylation status of p38 MAPK and JNK increased at the end of limb immobilization but returned to control values at recovery day 3. Transient increases in phosphorylation and in protein concentration occurred during recovery of soleus muscle mass. Phosphorylation of Akt, p70S6k, and signal transducer and activator of transcription 3 (STAT3) peaked on recovery day 3 compared with day 0. Glycogen synthase kinase (GSK)-3beta phosphorylation was increased on the sixth and fifteenth recovery day. In addition, transient peaks in seven protein concentrations occurred at different times of recovery: STAT3, calcineurin A (CaNA), CaNB, and beta4E-BP1 protein concentrations peaked on the third recovery day; p70S6k, STAT3, Akt, and GSK3-beta peaked on the sixth recovery day; and GSK3-beta peaked on the fifteenth recovery day. The apexes of STAT3 and GSK3-beta protein concentrations remained elevated for two recovery time points. Thus the time course of increase in molecules of signaling pathways differed as the young rat soleus muscle regrew from an atrophied state.

Alzheimer’s disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell–cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

. Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle. Physiol Genomics 15: 34-43, 2003. First published July 29, 2003 10.1152/physiolgenomics.00040.2003.-The direction of change in skeletal muscle mass differs between young and old individuals, growing in young animals and atrophying in old animals. The purpose of the experiment was to develop a statistically conservative list of genes whose expression differed significantly between young growing and old atrophying (sarcopenic) skeletal muscles, which may be contributing to physical frailty. Gene expression levels of Ͼ24,000 transcripts were determined in soleus muscle samples from young (3-4 mo) and old (30-31 mo) rats. Agerelated differences were determined using a Student's t-test (␣ of 0.05) with a Bonferroni adjustment, which yielded 682 probe sets that differed significantly between young (n ϭ 25) and old (n ϭ 20) animals. Of 347 total decreases in aged/ sarcopenic muscle relative to young muscles, 199 were functionally identified; the major theme being that 24% had a biological role in the extracellular matrix and cell adhesion. Three themes were observed from 213 of the 335 total increases in sarcopenic muscles whose functions were documented in databases: 1) 14% are involved in immune response; 2) 9% play a role in proteolysis, ubiquitin-dependent degradation, and proteasome components; and 3) 7% act in stress/antioxidant responses. A total of 270 differentially expressed genes and ESTs had unknown/unclear functions. By decreasing the sample sizes of young and old animals from 25 ϫ 20 to 15 ϫ 15, 10 ϫ 10, and 5 ϫ 5 observations, we observed 682, 331, 73, and 3 statistically different mRNAs, respectively. Use of large sample size and a Bonferroni multiple testing adjustment in combination yielded increased statistical power, while protecting against false positives. Finally, multiple mRNAs that differ between young growing and old, sarcopenic muscles were identified and may highlight new candidate mechanisms that regulate skeletal muscle mass during sarcopenia.