3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.

PI3K activation prevents Aß42-induced synapse loss and favors insoluble amyloid deposit formation.

Excess of Aß42 peptide is considered a hallmark of the disease. Here we express the human Aß42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Aß42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the coexpression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and thioflavin-positive amyloid deposits. Mechanistically, we suggest that Aß42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments, we further analyze Aß42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Aß42 aggregation into large insoluble deposits is reproduced. Finally, we show that the Aß42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.

Role of Tau Protein in Remodeling of Circadian Neuronal Circuits and Sleep.

Multiple neurological, physiological, and behavioral functions are synchronized by circadian clocks into daily rhythms. Neurodegenerative diseases such as Alzheimer's disease and related tauopathies are associated with a decay of circadian rhythms, disruption of sleep patterns, and impaired cognitive function but the mechanisms underlying these alterations are still unclear. Traditional approaches in neurodegeneration research have focused on understanding how pathology impinges on circadian function. Since in Alzheimer's disease and related tauopathies tau proteostasis is compromised, here we sought to understand the role of tau protein in neuronal circadian biology and related behavior. Considering molecular mechanisms underlying circadian rhythms are conserved from Drosophila to humans, here we took advantage of a recently developed tau-deficient Drosophila line to show that loss of tau promotes dysregulation of daily circadian rhythms and sleep patterns. Strikingly, tau deficiency dysregulates the structural plasticity of the small ventral lateral circadian pacemaker neurons by disrupting the temporal cytoskeletal remodeling of its dorsal axonal projections and by inducing a slight increase in the cytoplasmic accumulation of core clock proteins. Taken together, these results suggest that loss of tau function participates in the regulation of circadian rhythms by modulating the correct operation and connectivity of core circadian networks and related behavior.

Amyloid ß42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K.

The human Aß42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aß42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aß42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR) of adult flies at selected ages post Aß42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aß42 toxicity can be suppressed by the joint overexpression of PI3K.

Aberrant Wnt signaling: a special focus in CNS diseases.

Wnt signals regulate cell proliferation, migration and differentiation during development, as well as synaptic transmission and plasticity in the adult brain. Abnormal Wnt signaling is central to a number of brain pathologies. We review here, the significance of this pathway focused in the contribution of the most frequent alterations in receptors, secretable modulators and downstream targets in Alzheimer's disease (AD) and Glioblastoma (GBM). ß-catenin and GSK3 levels are pivotal in the neurodegeneration associated to AD contributing to memory deficits, tau phosphorylation, increased ß-amyloid production and modulation of Apolipoprotein E in the brain. In consequence, ß-catenin and GSK3 are targets for potential treatments in AD. Also, Wnt pathway components and secreted molecules interfering with this signaling contribute to the progression of tumoral cells. Wnt pathway activation is a bad prognosis in brain cancer; however, mutations in WNT or Frizzled (FZD) genes do not account for the cases of GBM. Instead, recent studies indicate that epigenetic modifications contribute to the development of GBMs opening novel strategies to study GBM progression.

Drosophila enhancer-Gal4 lines show ectopic expression during development.

In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers (elav, D42/Toll-6, OK6/RapGAP1) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal (Sxl) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used.

Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe.

We have previously shown that driving PI3K levels up or down leads to increases or reductions in the number of synapses, respectively. Using these tools to assay their behavioral effects in Drosophila melanogaster, we showed that a loss of synapses in two sets of local interneurons, GH298 and krasavietz, leads to olfaction changes toward attraction or repulsion, while the simultaneous manipulation of both sets of neurons restored normal olfactory indexes. We show here that olfactory central adaptation also requires the equilibrated changes in both sets of local interneurons. The same genetic manipulations directed to projection (GH146) or mushroom body (201Y, MB247) neurons did not affect adaptation. Also, we show that the equilibrium is a requirement for the glomerulus-specific size changes which are a morphological signature of central adaptation. Since the two sets of local neurons are mostly, although not exclusively, inhibitory (GH298) and excitatory (krasavietz), we interpret that the normal phenomena of sensory perception, measured as an olfactory index, and central adaptation rely on an inhibition/excitation ratio.

Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: a potential therapeutic role.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease for which there exist no therapies without undesired side effects. Thus, the establishment of less toxic treatments is an ongoing challenge. Nowadays, research on medicinal plants has been attracting much attention, since screening of its active principles could prove useful in identification of safe and innovative pharmaceutical molecules. In this study we investigated the therapeutic effect of oleanolic acid (OA) a plant-derived triterpene with potent anti-inflammatory and immunomodulatory activities, whose actions on CNS diseases remain far from completely characterized. We focussed on the potential therapeutic effect of oleanolic acid (OA) on an accepted experimental model of MS, the experimental autoimmune encephalomyelitis (EAE). We have found that OA treatment, before or at the early onset of EAE, ameliorates neurological signs of EAE-mice. These beneficial effects of OA seem to be associated with a reduction of blood-brain barrier leakage and lower infiltration of inflammatory cells within the CNS, as well as with its modulatory role in Th1/Th2 polarization: inhibition of proinflammatory cytokines and chemokines, and stimulation of anti-inflammatory ones. Moreover, EAE-animals that were treated with OA had lower levels of anti-MOG antibodies than untreated EAE-mice. Our findings show that the administration of the natural triterpenoid OA reduces and limits the severity and development of EAE. Therefore, OA therapy might be of clinical interest for human MS and other Th1 cell-mediated inflammatory diseases.

Secreted phospholipase A2-IIA modulates key regulators of proliferation on astrocytoma cells.

Human group IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA(2)-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA(2)-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA(2)-IIA-induced cell proliferation. We found that sPLA(2)-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA(2)-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA(2)-IIA treatment, effect that was abrogated by N-acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N-acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA(2)-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.