TrkB signaling directs the incorporation of newly generated periglomerular cells in the adult olfactory bulb.

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when inte-gration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-was found to be responsible for spine formation. Further-more, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB. \ua9 2013 the authors

Spatiotemporal control of mitochondrial network dynamics in astroglial cells

Mitochondria are increasingly recognized for playing important roles in regulating the evolving metabolic state of mammalian cells. This is particularly true for nerve cells, as dysregulation of mitochondrial dynamics are invariably associated with a number of neuropathies. Accumulating evidence now reveals that changes in mitochondrial dynamics and structure may play equally important roles also in the cell biology of astroglial cells. Astroglial cells display a significant heterogeneity in their morphology and specialized functions across the different brain regions, however besides fundamental differences they seem to share a surprisingly complex meshwork of mitochondria, which is highly suggestive of tightly regulated mechanisms that contribute to maintain this unique architecture. Here, we summarize recent work performed in astrocytes in situ indicating that this may indeed be the case, with astrocytic mitochondrial networks shown to experience rapid dynamic changes in response to defined external cues. Although the mechanisms underlying this degree of mitochondrial re-shaping are far from being understood, recent data suggest that they may contribute to demarcate astrocyte territories undergoing key signalling and metabolic functions

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Synopsis image Glial cell line derived neurotrophic factor (GDNF) improves survival in toxin-models of Parkinson's disease and is currently undergoing clinical development, however the protective mechanism is elusive. This study provides evidence that the GDNF receptor Ret rescues defects of a genetic Parkinson model and proposes a new mechanism-of-action. Active Ret overexpression rescues muscle degeneration and mitochondrial morphology in muscles and dopamine neurons in Pink1 mutant Drosophila. In human neuroblastoma cells, GDNF treatment rescues mitochondrial fragmentation caused by Pink1 knockdown. Ret signaling improves mitochondrial respiration and activity of complex I, providing a potential novel mechanism for the protective effect of GDNF/Ret. Abstract Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret(MEN)(2B)) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret(MEN)(2B) significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD

Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 \ub5M sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular disease

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Hormesis and cardioprotection

Oxidative stress has been reported to play a causative role in cardiovascular diseases. Although reactive oxygen species (ROS) are known to enhance myocardial ischemia-reperfusion injury (Morihira et al, 2005), it has been demonstrated that a short-timed ischemia, also called preconditioning, may exert a protective role, preventing thus from a subsequent prolonged stress. Several factors have been reported to mediate preconditioning mechanism, such as blockade of reduction in mitochondrial membrane potential or modulation of multiple genes and signalling pathways (Tang et al, 2005). Anyway, the apparent paradox of preconditioning is strictly connected with a biphasic process known as \u201chormesis\u201d. Hormesis is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition: as a consequence, the same stressor that at higher doses appears toxic, at lower ones leads to beneficial effects (Calabrese and Baldwin, 2002). In this way, preconditioning can be considered as an hormetic response as well. The aim of this work is to investigate the probable protective role of preconditioning on oxidative stress in cultured cardiomyocytes and its effects on antioxidant enzymatic machinery, in order to enlighten the mechanism of protection and the targets involved. In our study we used H2O2 as stressor, as it is well documented that this molecule is capable of adaptative responses similar to those induced by ischemic preconditioning. Materials and methods. Cultured rat neonatal cardiomyocytes were prepared and grown until confluence as previously reported (Hrelia et al., 2002). Preconditiong was simulated exposing the cells to 100 \ub5M H2O2 for ten minutes and, 24 hours later, oxidative stress was induced with 100 \ub5M H2O2 for 30 minutes. Cell viability was evaluated by MTT assay. Apoptosis and necrosis analysis was assessed by cytofluorimetric assay using Guava Nexin Assay (Guava Technologies). Enzymatic activities of glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPX), thioredoxin reductase (TR), NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT) and superoxide dismutase (SOD) were determined by spectrophotometric methods. Results and discussion. MTT results indicate a significant protective effect of preconditioning against oxidative stress, preconditioning with 100 \ub5M H2O2 led to a complete protection against subsequent peroxide induced-injury. In agreement with MTT data, cytofluorimetric assay showed that preconditioning was able to reduce necrotic and apoptotic populations and increased viable cells in comparison to oxidative stress only. Preconditioning was able to increase GR, NQO1, TR and CAT activities, while it did not influence GPX, GST, and SOD activities. So the preconditioning protective effect may be ascribed to an increased cell ability to detoxify ROS by increasing the activities of fundamental antioxidant/phase II enzymes. As all these enzymes are regulated through the Keap1-Nrf2-antioxidant responsive element signalling pathway, their unlinked regulation underpins the hypothesis that preconditioning could modulate these enzymes through different mechanisms. Supported by Fondazione del Monte di Bologna e Ravenna

Omega-3 fatty acid and antioxidant enriched liposomes for nasal administration of anti-Alzheimer drugs

The purpose of this work was the formulation of liposomes for nasal administration of tacrine\ub7HCl. This route has many advantages, in fact it is possible to convey the drug directly to the CNS, through the olfactory bulb. Part of tacrine administered can also be absorbed into the systemic circulation avoiding hepatic first pass effect. Liposomes were formulated with ingredients that have a beneficial effect against the disease. In particular, the cholesterol was replaced with α-tocopherol, and phospholipids were enriched with polyunsaturated fatty acids (DHA, EPA α-linolenic acid). This formulative approach allowed us to obtain 100% active liposomes against symptoms of Alzheimer's disease