• The control and effects of Foxo1 in skeletal muscle

      Schachter, Tova Neustadt; Schneider, Martin F. (2012)
      In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins which mediate muscle atrophy, making the nuclear concentration and nuclear/cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the IGF-1/PI3K/Akt pathway, to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway, and to explore the effects of Foxo1 on contraction of muscle fibers. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier CRM1 by Leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear/cytoplasmic shuttling of Foxo1 under control conditions prior to LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles about 20 fold more slowly than Foxo1-GFP. Fibers expressing Foxo1-GFP exhibit an inability to contract, abnormal propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared to fibers expressing GFP alone. Evaluation of the T-tubule system, the membranous system involved in the radial and longitudinal propagation of the action potential, using a membrane fluorescent dye, revealed an intact T-tubule network in fibers over-expressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment in Foxo1-GFP fibers induced recovery of normal calcium transients, indicating that Foxo1 translocation affects the expression of proteins involved in the generation and/or propagation of action potentials. A decrease in Nav1.4 expression in fibers overexpressing Foxo1 was also observed in the absence of IGF-1. We conclude that overactivity of Foxo1 prevents the normal muscle responses to electrical stimulation by decreasing expression of Nav1.4 and possibly other means. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions, as well as the effects of Foxo1 on the electrophysiology of muscle.
    • Differential FOXO1 Localization in SLE and Healthy Human Lymphocyte Subsets

      Hritzo, Molly K.; Golding, Amit; 0000-0001-9614-732X (2018)
      Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by elevated levels of circulating autoantibodies and multi-organ damage. Although SLE is a highly heterogeneous disease, one factor unifies it: lymphocyte hyperactivity driving immunopathogenesis. This involves CD4 helper T cells potentiating autoreactive B cells to produce pathogenic autoantibodies. In healthy individuals, lymphocyte activation is a closely regulated kinetic process controlled by key transcription factors (TF) signaling downstream of the T cell (TCR) and B cell receptor (BCR). Forkhead box O1 (FOXO1) is one such TF that integrates activation and differentiation signals in human lymphocytes. When active, it remains in the nucleus, but upon Akt phosphorylation downstream of TCR or BCR signaling, FOXO1 is inactivated and shuttles to the cytoplasm, linking FOXO1 localization to function. In SLE, both T and B cells are hyperactive, and respond more quickly and strongly to antigen, producing a disproportionate inflammatory response. Thus, we hypothesize that SLE lymphocytes will have altered FOXO1 localization, reflecting altered lymphocyte activation. To address this hypothesis, we first developed a method of examining dynamic native FOXO1 localization in human peripheral lymphocyte subsets using imaging flow cytometry (IFC). IFC combines the quantitative power of flow cytometry with the qualitative images of microscopy and can be performed with many fewer cells than are needed for the more traditional methods. We demonstrated that we can visualize native FOXO1 and detect significant kinetic differences in localization within user-defined subsets of HuT102 cells, a human CD4 T cell line with baseline nuclear FOXO1, as well as primary peripheral human T and B cells. We then used IFC to compare FOXO1 localization in SLE and healthy donor lymphocytes. Interestingly, we found that most T and B cell subsets have nuclear FOXO1 localization in both health and SLE. However, FOXO1 is significantly more cytoplasmic in SLE double negative (DN) atypical memory B cells. Based on our findings, we propose a model by which these DN B cells are highly active in disease flares and may serve as a death-resistant reservoir of autoreactive cells. Future experiments will be aimed at elucidating at how these cells persist in the periphery.
    • Green Tea (EGCG), Insulin, IGF-1 Suppression of Atrophy Associated Transcription Factor Foxo1 Activity in Skeletal Muscle and Mathematical Modeling of Nuclear Influx Efflux of Foxo1 in Skeletal Muscle

      Wimmer, Robert; Schneider, Martin F. (2014)
      Preventing, slowing and reversing skeletal muscle atrophy offers the potential to substantially improve the quality of life for our increasingly aging population. In addition to the primary function of force generation for movement, skeletal muscle plays an equally important role in maintenance of homeostasis through regulation of metabolic critical organic compounds such as amino acids, growth hormones, fatty acids, and glucose. Disruption of the body's homeostasis during diseases such as cancer, renal failure, sepsis, or diabetes leads to significant skeletal muscle atrophy. Our study utilizes over-expression of a skeletal muscle atrophy associated transcription factor, Foxo1, tagged with a green fluorescent protein (GFP) to provide a visual indication where within the muscle fiber (Nucleus or Cytoplasm) Foxo1 is located. This provides an indication of the activation status of the Foxo1 by comparing nuclear to cytoplasmic ratios with time. A two state mathematical model was created to account for the observed Foxo1-GFP nuclear concentration time course and presented here in Chapter 2. This model made two interesting observations, one implying that the kinase Akt not the predominant enzyme that phosphorylates Foxo1 in the nucleus prior to Foxo1 nuclear efflux. The second indicates that in the presence of IGF, intra-nuclear Akt activity, or possibly the activity of another kinase, has become strongly activated with the presence of IGF within the muscle fiber nuclei. EGCG is found in green tea, the most popularly consumed beverage in the world after water. Presented in Chapter 3, we show results of reduced Foxo1 activation induced by EGCG which are similar to the effects of well established endogenous growth hormones, IGF-1 and insulin. Interestingly EGCG appears to have its effect at least partially via parallel signaling pathways that are independent of IGF-1's (and insulin's) downstream PI3K/Akt/Foxo1 signaling axis. Future work to further understand EGCG's parallel signaling pathways could have implications both in slowing muscle atrophy as it relates to Foxo1 or, more broadly in providing a clinical parallel pathway to IGF-1 and/or insulin.