Faculty Bibliography

2-hydroxypropyl-beta-cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo-lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann-Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild-type mice and TgCRND8 mice - an Alzheimer's Disease (AD) model exhibiting beta-amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14-day intracerebroventricular administration of CYCLO to 8 month old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and Abeta-immunoreactivity without detectably altering amyloid precursor protein processing or extracellular Abeta/beta-amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome-lysosome fusion as evidenced by accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

Necroptosis is a caspase 8-independent cell death that requires co-activation of receptor-interacting protein (RIP) 1 and RIP 3 kinases. The necrosome is a complex consisting of RIP1, RIP3 and Fas-associated protein with death domain (FADD) leading to activation of the pseudokinase mixed lineage kinase like (MLKL) followed by a rapid plasma membrane rupture and inflammatory response through the release of damage-associated molecular patterns (DAMPs) and cytokines. The necrosome has been shown to be relevant in multiple tumor types, including pancreatic adenocarcinoma, melanoma and several hematological malignancies. Preclinical data suggest that targeting this complex can have differential impact on tumor progression and that the effect of necroptosis on oncogenesis is cell type- and context-dependent. The emerging data suggest that targeting the necrosome may lead to immunogenic reprogramming in the tumor microenvironment in multiple tumors and that combining therapies targeting the necrosome with either conventional chemotherapy or immunotherapy may have beneficial effects. Thus, understanding the interplay of necroptotic cell death, transformed cells, and the immune system may enable the development of novel therapeutic approaches.

Purpose/Aim of the study: Poor cardiovascular health, including obesity and altered lipid profiles at mid-life, are linked to increased risk of Alzheimer's disease (AD). The biological mechanisms linking cardiovascular health and cognitive function are unclear though are likely to be multifactorial. This study examined the association between various lipoproteins and cognitive functioning in ageing women.

INTRODUCTION: According to a recent study from the Center of Disease Control and Prevention, 1 in every 10 Americans aged 12 and older reported chronic use of antidepressants. Chronic use of serotonin re-uptake inhibitors (SSRI) has been linked to impaired bone mineral accrual during skeletal development and osteoporosis [1,2]. We investigated the effect of fluoxetine, the most commonly prescribed SSRI in the U.S., on the complex program of bone regeneration in two disparate models of fracture repair in mice, followed by a thorough assessment of the in vitro mineralization capacity of primary osteoprogenitor cells (OPCs). We hypothesized that fluoxetine exerts a negative effect on osteoblast proliferation and differentiation during the process of fracture repair, resulting in a less mineralized and weaker bony callus. METHODS: Twelve week-old C57BL/6J mice were used following the IACUC guidelines at our institution. Fluoxetine was delivered in the drinking water at 10 mg/kg/day dose during the 3 weeks before surgeries to simulate chronic SSRI use [3]. Bone fracture repair through endochondral ossification was analyzed using a well-established femur fracture model stabilized with an intramedullary rod. Fracture callus was examined at 14 and 28 days. Intramembranous ossification was analyzed using a 1-mm monocortical tibial defect model. Here, injuries were allowed to heal for 7 or 14 days. Samples were subjected to microCT analysis, histomorphometry, TRAP and ALP histochemistry and immunolabeling for osteocalcin and runx2. A set of fractured femurs at d28 was subjected to 4-point biomechanical bending tests. All mice were continuously treated with fluoxetine during the repair period, except for a group of mice in which we aimed at understanding how discontinuation of the SSRI at the time of fracture would affect fracture healing (Fig. 1A). For the in vitro studies, bone marrow stromal cells were cultured in growth media alone or in presence of 5, 10 or 20 microM fluoxetine, with and without serotonin. Cell proliferation was measured using a BrdU colorimetric assay and apoptotic cells were detected by TUNEL labeling. bMSCs were also cultured in osteogenic differentiation media alone or with the aforementioned fluoxetine concentrations. Mineralization activity was analyzed by alizarin red staining and ALP activity and the expression of osteogenic markers was evaluated by qRT-PCR. An additional set of in vitro experiments was carried out with serotonin supplementation at 50mM in growth media or osteogenic media. Cell proliferation and osteogenic differentiation were examined. Student's t test with Holm-Sidak correction were used to quantify differences described in this study. Error bars represent standard deviation. An asterisk symbol (*) denotes a p value of less than 0.05. RESULTS: Fluoxetine-treated mice developed a normal cartilaginous callus at 14 days after fracture. At 28 days, the fluoxetine-treated animals demonstrated a significantly smaller and biomechanically weaker bony callus (Fig. 1B). In order to further dissect the mechanism that resulted in a smaller osseous regenerate, we studied the healing process of monocortical tibial defects as an intramembranous model of bone healing, which confirmed a direct effect of fluoxetine on osteoblast differentiation and mineralization. In vitro studies established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. Interestingly, histologic and microCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus (Fig. 1). DISCUSSION: In summary, our current study shows that chronic fluoxetine treatment negatively affects bone healing by inhibiting proliferation, osteoblast differentiation and mineralization. Data from this study and others provide strong evidence that chronic SSRI use leads to osteoporosis, which is associated with an increased fracture risk. In a translation arm of our study, we aimed at studying the effect of fluoxetine cessation at the time of fracture. In this group, we surprisingly encountered the consistent formation of non-unions with persistent fibrous tissue interposition. Further studies are now focusing at understanding this intriguing finding. (Figure Presented)

Pancreatic cancer is a devastating disease and ranks as the third most common cause of cancer-related death. Like many cancers, there has been increased interest in the role of the immune system in the progression and development of pancreatic cancer. In particular, immunosuppression within the tumor microenvironment (TME) is thought to impair the host's antitumor response. In this article, we review myeloid-derived suppressor cells and their contribution to this immunosuppression within the pancreatic TME.

Epidemiological and animal studies show that deleterious maternal environments predispose aging offspring to metabolic disorders and type 2 diabetes. Young progenies in a rat model of maternal low-protein (LP) diet are normoglycemic despite collapsed insulin secretion. However, without further worsening of the insulin secretion defect, glucose homeostasis deteriorates in aging LP descendants. Here we report that normoglycemic and insulinopenic 3-month-old LP progeny shows increased body temperature and energy dissipation in association with enhanced brown adipose tissue (BAT) activity. In addition, it is protected against a cold challenge and high-fat diet (HFD)-induced obesity with associated insulin resistance and hyperglycemia. Surgical BAT ablation in 3-month-old LP offspring normalizes body temperature and causes postprandial hyperglycemia. At 10 months, BAT activity declines in LP progeny with the appearance of reduced protection to HFD-induced obesity; at 18 months, LP progeny displays a BAT activity comparable to control offspring and insulin resistance and hyperglycemia occur. Together our findings identify BAT as a decisive physiological determinant of the onset of metabolic dysregulation in offspring predisposed to altered beta-cell function and hyperglycemia and place it as a critical regulator of fetal programming of adult metabolic disease.

INTRODUCTION: All tissues are affected by aging, but diseases that weaken the skeleton constitute the most prevalent chronic impairment in the United States. Although skeletal diseases and conditions are seldom fatal, they can significantly compromise function and diminish quality of life. Perhaps most importantly, age-related changes in skeletal health can be traced back to a decline in both the number and function of osteoprogenitor cells (OPCs). However, the cause for the decline in both the number and function of OPCs is not well understood. Chronic inflammation in the elderly (inflamm-aging) is thought to be a major contributor to this decline in the regenerative capacity of many tissues, including the skeleton. In contrast to a well-balanced inflammatory response after trauma, which is crucial for successful bone repair, chronic unbalanced elevation of pro-inflammatory cytokines has been shown to inhibit regeneration in a variety of tissues. We hypothesize that inflamm-aging is the major cause for the decline in OPC number and dysfunction in elderly patients and that this decline in OPC number and dysfunction can be halted by treatment with an anti-inflammatory drug. METHODS: Young, 12 week-old and aged, 52 week-old C57BL/6J were used following the IACUC guidelines at our institution. Aged animals were randomly distributed into a no-treatment (n=5) and a treatment group (n=5). Animals in the no-treatment group received regular drinking water, while animals in the treatment group received sodium salicylate water (12mg/day) for 8 weeks. The inflammatory status of young and aged untreated and treated mice was assessed using a multiplex platform screening for multiple pro- and anti-inflammatory cytokines, and utilizing qRT-PCR for IL-1, IL-6, NF-kappaB, TNF-alpha. FACS analysis using the LepR as a marker for osteogenic precursor cells was employed to identify the effect of chronic low-level inflammation on progenitor cell number. In addition, bMSCs were harvested from femurs and tibia from young and aged untreated and treated mice and cultured in growth media and osteogenic media. Cell proliferation and osteogenic differentiation (qRT-PCR for col 1, runx2, osx and oc, and alizarin red and alkaline phosphatase staining) were assessed in vitro. Results are presented in the form of mean +/- standard deviation, with N equal to the number of samples analyzed. Two-tailed Student's t-tests were used to determine significant differences between data sets that are normally distributed. For non-normally distributed data sets, Mann-Whitney U test was used. Significance was attained at p < 0.05 and all statistical analyses were performed with Graphpad Prism software (GraphPad Software, San Diego, California). RESULTS: First, we set out to identify age-related chronic inflammation in mice. We analyzed blood by multiplex analysis and tibial and femoral bone marrow by qRT-PCR for pro-inflammatory markers. Both analyses revealed an increase in pro-inflammatory and a decrease in anti-inflammatory cytokines in aged animals, confirming the presence of inflamm-aging (Fig. 1A). Next, we aimed at understanding how aging effects osteoprogenitor cell number using flow cytometry. We harvested cells from young and aged mice, removed red blood cells, and then stained with antibodies to CD31, CD45, Ter-119 and LepR. Cell sorting was performed and CD31^-CD45^-Ter^-119^-LepR+ cells were isolated and quantified. Flow cytometry analysis revealed that 0.38% of cells from 12 week old mice were LepR⁺ osteoprogenitor cells, confirming the findings published by Zhou et al. Analysis of the cells from 52 week-old mice revealed a significant decrease in number to 0.017% of bone marrow cells (Fig. 1B). Next, we had to establish that an 8-week course of sodium salicylate successfully represses chronic inflammation. In response to NSAID treatment, the expression level of IL-10 significantly increased above the level of the juvenile animals, while the NF-kappaB, TNFalpha and Cox-2 levels returned to baseline (Fig. 1A). This experiment served as a proof-of-principle that mice exhibit inflamm-aging and that this inflammatory state can be suppressed by administration of an NSAID. Having established that our dosing of the systemic NSAID suppresses chronic ageinduced inflammation, we assessed whether OPC frequency changes as a result of suppressed inflamm-aging. Bone marrow from 3 month-old, 12 month-old and 12 monthold NSAID-treated animals was subjected to flow cytometry. OPC frequency declined with aging, however, after an 8 week course of NSAID treatment, we noticed a two-fold increase in LepR⁺ OPCs within the bone marrow (Fig. 1B). We then aimed at testing whether NSAID treatment of aged mice resulted in a restoration of the osteogenic potential of this OPC population. Quantitative RT-PCR of the bone marrow of sodium salicylate-treated mice showed an increase in osteogenic gene expression (osx, oc and alkaline phosphatase) compared to untreated aged mice. The expression level of osx reached that of young, 3 month-old mice, while oc and ALP expression levels were significantly higher than those of juvenile animals (Fig. 1C). In order to further characterize this increase in osteogenic potential, we harvested MSCs from young and aged treated and untreated animals, plated them in vitro and then subjected them to osteogenic differentiation media. Mineralization assays and expression analysis of osx, oc and ALP showed decreased osteogenesis of aged cells, while treatment with sodium salicylate recovered this decline and resulted in restoration of the osteogenic potential (Fig. 1D). DISCUSSION: These experiments demonstrate for the first time that age-related chronic inflammation is responsible for the decreased proliferative and osteogenic potential of aged OPCs and that this process is reversible by anti-inflammatory treatment. The findings from this study may have a profound translational impact: If we could restore the regenerative potential of the aged skeleton by treating age-related inflammation, then theoretically, we may have a tool at hand to improve the healing process of osteoporotic fracture patients. (Figure Presented)