Speech Communication

Contains reports on five research projects.U. S. Air Force (Electronic Systems Division) under Contract AF 19(628)-3325National Science Foundation (Grant GP-Z495)National Institutes of Health (Grant MH-04737-04)National Institutes of Health (Grant NB-0433Z-02)National Aeronautics and Space Administration (Grant NsG-496

MicroRNAs control neurobehavioral development and function in zebrafish

microRNAs (miRNAs) have emerged as regulators of a broad spectrum of neurodevelopmental processes, including brain morphogenesis, neuronal differentiation, and survival. While the role of miRNAs in establishing and maintaining the developing nervous system is widely appreciated, the developmental neurobehavioral role of miRNAs has yet to be defined. Here we show that transient disruption of brain morphogenesis by ethanol exposure results in behavioral hyperactivity in larval zebrafish challenged with changes in lighting conditions. Aberrations in swimming activity persist in juveniles that were developmentally exposed to ethanol. During early neurogenesis, multiple gene expression profiling studies revealed widespread changes in mRNA and miRNA abundance in ethanol-exposed embryos. Consistent with a role for miRNAs in neurobehavioral development, target prediction analyses identified multiple miRNAs misexpressed in the ethanol-exposed cohorts that were also predicted to target inversely expressed transcripts known to influence brain morphogenesis. In vivo knockdown of miR9/9* or miR-153c persistently phenocopied the effect of ethanol on larval and juvenile swimming behavior. Structural analyses performed on adults showed that repression of miR-153c during development impacts craniofacial skeletal development. Together, these data support an integral role for miRNAs in the establishment of vertebrate neurobehavioral and skeletal systems.-Tal, T. L., Franzosa, J. A., Tilton, S. C., Philbrick, K. A., Iwaniec, U. T., Turner, R. T., Waters, K. M., Tanguay, R. L. MicroRNAs control neurobehavioral development and function in zebrafish. FASEB J. 26, 1452-1461 (2012). www.fasebj.orgKeywords: Brain, Morphogenesis, Alcohol, Conditional loss, Osteoblast differentiation, Gene expression, Microarray data, Neural progenitors, Ethanol exposure, Larval zebrafis

Polyethylene particles inserted over calvarium induce cancellous bone loss in femur in female mice

Focal bone resorption (osteolysis) induced by wear particles contributes to long-term orthopedic joint failure. However, the impact of focal osteolysis on remote skeletal sites has received less attention. The goal of this study was to determine the effects of polyethylene particles placed over calvaria on representative axial and appendicular skeletal sites in female mice. Because recent work has identified housing temperature as an important biological variable in mice, response to particle treatment was measured in animals housed at room (22 °C) and thermoneutral (32 °C) temperature. Osteolysis was evident in skeletal tissue adjacent to particle insertion. In addition, cancellous bone loss was observed in distal femur metaphysis. The bone loss was associated with lower osteoblast-lined perimeter and lower mineralizing perimeter in distal femur, lower osteocalcin gene expression in tibia, and lower serum osteocalcin, suggesting the response was due, at least in part, to reduced bone formation. Mild cold stress induced by sub-thermoneutral housing resulted in cancellous bone loss in distal femur and lumbar vertebra but did not influence skeletal response to particles. In summary, the results indicate that focal inflammation induced by polyethylene particles has the potential to result in systemic bone loss. This is significant because bone loss is a risk factor for fracture. Keywords: Osteolysis, Bone resorption, Bone formation, Osteoporosi

Hypothalamic Leptin Gene Therapy Reduces Bone Marrow Adiposity in ob/ob Mice Fed Regular and High Fat Diets

Low bone mass is often associated with increased bone marrow adiposity. Since osteoblasts and adipocytes are derived from the same mesenchymal stem cell progenitor, adipocyte formation may increase at the expense of osteoblast formation. Leptin is an adipocyte-derived hormone known to regulate energy and bone metabolism. Genetic (e.g., leptin deficiency) and high fat diet-induced (e.g., leptin resistance) obesity are associated with increased marrow adipose tissue (MAT) and reduced bone formation. Short-duration studies suggest that leptin treatment reduces MAT and increases bone formation in leptin-deficient ob/ob mice fed a regular diet. Here, we determined the long-duration impact of increased hypothalamic leptin on marrow adipocytes and osteoblasts in ob/ob mice using recombinant adeno-associated virus (rAAV) gene therapy. In a first study, eight- to ten-week-old male ob/ob mice were randomized into 4 groups: (1) untreated, (2) rAAV-Lep, (3) rAAV-green fluorescent protein (rAAV-GFP), or (4) pair-fed to rAAV-Lep. For vector administration, mice were placed in a Kopf stereotaxic apparatus, and injected intracerebroventricularly with either rAAV-Lep or rAAV-GFP (9 × 107 particles in 1.5 µl). The mice were maintained for 30 weeks following vector administration. In a second study, the impact of increased hypothalamic leptin levels on MAT was determined in mice fed high fat diets. Eight- to ten-week-old male ob/ob mice were randomized into 2 groups and treated with either rAAV-Lep or rAAV-GFP. At 7 weeks post-vector administration, half the mice in each group were switched to a high fat diet for 8 weeks. Wild type (WT) controls included age-matched mice fed regular or high fat diet. Hypothalamic leptin gene therapy increased osteoblast perimeter and osteoclast perimeter with minor change in cancellous bone architecture. The gene therapy decreased MAT levels in ob/ob mice fed regular or high fat diet to values similar to WT mice fed regular diet. These findings suggest that leptin plays an important role in regulating the differentiation of mesenchymal stem cells to adipocytes and osteoblasts. Furthermore, leptin resistance may play a role in the switch from osteoblastogenesis to adipogenesis associated with obesity. However, the results also illustrate that reducing MAT does not necessarily result in increased bone mass

DataSheet_1_Leptin and environmental temperature as determinants of bone marrow adiposity in female mice.pdf

Bone marrow adipose tissue (BMAT) levels are higher in distal femur metaphysis of female mice housed at thermoneutral (32°C) than in mice housed at 22°C, as are abdominal white adipose tissue (WAT) mass, and serum leptin levels. We performed two experiments to explore the role of increased leptin in temperature-enhanced accrual of BMAT. First, we supplemented 6-week-old female C57BL/6J (B6) mice with leptin for 2 weeks at 10 µg/d using a subcutaneously implanted osmotic pump. Controls consisted of ad libitum (ad lib) fed mice and mice pair fed to match food intake of leptin-supplemented mice. The mice were maintained at 32°C for the duration of treatment. At necropsy, serum leptin in leptin-supplemented mice did not differ from ad lib mice, suggesting suppression of endogenous leptin production. In support, Ucp1 expression in BAT, percent body fat, and abdominal WAT mass were lower in leptin-supplemented mice. Leptin-supplemented mice also had lower BMAT and higher bone formation in distal femur metaphysis compared to the ad lib group, changes not replicated by pair-feeding. In the second experiment, BMAT response was evaluated in 6-week-old female B6 wild type (WT), leptin-deficient ob/ob and leptin-treated (0.3 μg/d) ob/ob mice housed at 32°C for the 2-week duration of the treatment. Compared to mice sacrificed at baseline (22°C), BMAT increased in ob/ob mice as well as WT mice, indicating a leptin independent response to increased temperature. However, infusion of ob/ob mice with leptin, at a dose rate having negligible effects on either energy metabolism or serum leptin levels, attenuated the increase in BMAT. In summary, increased housing temperature and increased leptin have independent but opposing effects on BMAT in mice.</p