A conserved role for sleep in supporting Spatial Learning in Drosophila.

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. Here we use the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a "thermal maze" consisting of a 5 × 5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains-Cs and 2U, validating our design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, we found that flies exhibited a dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, we find that Spatial Learning requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. Our results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.

Enhanced sleep reverses memory deficits and underlying pathology in Drosophila models of Alzheimer's disease.

To test the hypothesis that sleep can reverse cognitive impairment during Alzheimer's disease, we enhanced sleep in flies either co-expressing human amyloid precursor protein and Beta-secretase (APP:BACE), or in flies expressing human tau. The ubiquitous expression of APP:BACE or human tau disrupted sleep. The sleep deficits could be reversed and sleep could be enhanced when flies were administered the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Expressing APP:BACE disrupted both Short-term memory (STM) and Long-term memory (LTM) as assessed using Aversive Phototaxic Suppression (APS) and courtship conditioning. Flies expressing APP:BACE also showed reduced levels of the synaptic protein discs large (DLG). Enhancing sleep in memory-impaired APP:BACE flies fully restored both STM and LTM and restored DLG levels. Sleep also restored STM to flies expressing human tau. Using live-brain imaging of individual clock neurons expressing both tau and the cAMP sensor Epac1-camps, we found that tau disrupted cAMP signaling. Importantly, enhancing sleep in flies expressing human tau restored proper cAMP signaling. Thus, we demonstrate that sleep can be used as a therapeutic to reverse deficits that accrue during the expression of toxic peptides associated with Alzheimer's disease.

Sleep restores behavioral plasticity to Drosophila mutants.

Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

Neural stem cells-trends and advances.

For many years, accepted dogma held that brain is a static organ with no possibility of regeneration of cells in injured or diseased human brain. However, recent preclinical reports have shown regenerative potential of neural stem cells using various injury models. This has resulted in renewed hope for those suffering from spinal cord injury and neural damage. As the potential of stem cell therapy gained impact, these claims, in particular, led to widespread enthusiasm that acute and chronic injury of the nervous system would soon be a problem of the past. The devastation caused by injury or diseases of the brain and spinal cord led to wide premature acceptance that "neural stem cells (NSCs)" derived from embryonic, fetal or adult sources would soon be effective in reversing neural and spinal trauma. However, neural therapy with stem cells has not been realized to its fullest extent. Although, discrete population of regenerative stem cells seems to be present in specific areas of human brain, the function of these cells is unclear. However, similar cells in animals seem to play important role in postnatal growth as well as recovery of neural tissue from injury, anoxia, or disease.

Gamma radiation induces senescence in human adult mesenchymal stem cells from bone marrow and periodontal ligaments.

PURPOSE: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. METHODS: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co(60) and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. RESULTS: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21(Cip1/Waf1)) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16(INK4a)) and increased activity of senescence-associated ß-galactosidase. CONCLUSION: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.

Splenectomy influences homing of transplanted stem cells in bone marrow-ablated mice.

Cell mobilization, a process that influences circulation, margination, and finally, homing play key roles in the regeneration processes mediated by stem cells. Recent studies as well as prior studies from our group indicate an important role of the spleen in hematopoietic reconstitution, but to date the role of the spleen in hematopoietic reconstitution has been unclear and it has not been precisely documented in ablated animals. Therefore, we undertook the present study to define more closely the role of the spleen in hematopoietic reconstitution in lethally irradiated mice. After transplantation of irradiated mice with lacZ+ -marked lin- / CD117+ bone marrow cells, we compared splenectomized mice (T(S), splenectomy performed prior to irradiation) to nonsplenectomized, irradiated mice (T(N)) as well as to normal (unirradiated) mice. Impaired hematopoietic reconstitution was observed in T(S) mice. Splenectomy markedly altered the distribution of hematopoietic stem cells, as demonstrated by fluorescence-activated cell sorting analysis of endogenous CD117+ cells in the thymus and bone marrow of recipients. Cell engraftment was demonstrated by histochemical and polymerase chain reaction analyses of recipient tissues. These experiments demonstrated that in T(S) animals, transplanted hematopoietic stem cells mobilized to extravascular tissues, particularly the gastrointestinal tract. The number of donor cells in recipient tissues continued to increase for 30 days after transplantation with the highest numbers observed in the T(S) group. DNA marking analysis led to the conclusion that engrafted cells were not only integrated into recipient tissues but were also capable of performing complex cellular processes, including proliferation and repair. Our results are consistent with the novel possibility that cellular repair markedly affects stem cell regenerative functions and that repair is markedly influenced by the integrity and presence of organs not directly involved in specific tissue regeneration processes, particularly the spleen.

Irradiation of adult human dental pulp stem cells provokes activation of p53, cell cycle arrest, and senescence but not apoptosis.

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-ß-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.

Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging.

Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch's membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy.