Mouse embryonic stem cells as a discovery tool in neurobiology

Mouse ES cells can recapitulate, under suitable tissue culture conditions, early events in neurogenesis. As wildtype or genetically modified ES cells can be grown in unlimited quantities, their differentiation into neurons represents an attractive model for studying the function of genes involved in early development, such as those controlling neuronal specification and survival. A few years ago, our laboratory established a robust differentiation protocol leading to the generation of well-defined and virtually pure populations of Pax6-positive radial glial (RG) cells with a profile and developmental potential characteristic of Pax6-positive RG cells of the cortex. Like their in vivo counterparts, these progenitors generate homogeneous populations of glutamatergic neurons. In my thesis work, I first addressed the role of Pax6 in the generation, specification and developmental potential of RG cells, by analyzing the progeny of ES cells isolated from homozygote Pax6-mutant embryos. I found that while Pax6 is not required for the generation of neurogenic RG, it is both sufficient and necessary for specifying them into a glutamatergic lineage. RG cells lacking Pax6 express genes specifying an interneuron fate, like Mash1, and generate GABAergic inhibitory neurons. These cells die prematurely due to an aberrant over-expression of the neurotrophin receptor p75. I could verify these findings in the cortex of mutant embryos lacking Pax6. This work led to new insights as to the regulation of neuronal specification and survival during neurogenesis. In the second part of my thesis, I used this ES cell-based differentiation system to test any potential instructive roles of the 3 neurotrophin tyrosine kinase receptors TrkA, TrkB and TrkC, after recombining them into the neuron-specific mapt locus. This approach led to the surprising observations that TrkA and TrkC cause neuronal death when not activated by their neurotrophin ligands, whereas TrkB does not. Both the death inducing activity of TrkA and TrkC and the lack of death-inducing activity of TrkB were explained by differential distribution of these receptors with p75. The TrkA and TrkC-induced death involves their segregation together with p75 in lipid rafts, and the subsequent proteolysis of the latter signals to the apoptotic machinery. By contrast, TrkB is not recruited to lipid rafts and it does not result in p75 proteolysis. Subsequent analyses of TrkA and ngf mutants, as well as of embryos lacking both TrkA and p75 receptors confirmed the relevance of this novel death triggering mechanism during the development of the peripheral nervous system. These findings also point to a major, and so far un-described, difference in the way growth factors regulate the survival of neurons in the developing peripheral versus central nervous system. It is the receptors themselves that cause neurons to become growth factor dependent in the peripheral, but not in the central nervous system. Taken together, my results demonstrate that the differentiation of mouse embryonic stem cells into defined neuronal populations represents a useful tool allowing observations to be made that are relevant to the development of the nervous system

Calcium homeostasis in aging neurons

The nervous system becomes increasingly vulnerable to insults and prone to dysfunction during ageing. Age-related decline of neuronal function is manifested by the late onset of many neurodegenerative disorders, as well as by reduced signalling and processing capacity of individual neuron populations. Recent findings indicate that impairment of Ca2+ homeostasis underlies the increased susceptibility of neurons to damage, associated with the ageing process. However, the impact of ageing on Ca2+ homeostasis in neurons remains largely unknown. Here, we survey the molecular mechanisms that mediate neuronal Ca2+ homeostasis and discuss the impact of ageing on their efficacy. To address the question of how ageing impinges on Ca2+ homeostasis, we consider potential nodes through which mechanisms regulating Ca2+ levels interface with molecular pathways known to influence the process of ageing and senescent decline. Delineation of this crosstalk would facilitate the development of interventions aiming to fortify neurons against age-associated functional deterioration and death by augmenting Ca2+ homeostasis

Macroautophagy and normal aging of the nervous system: Lessons from animal models.

Aging represents a cumulative form of cellular stress, which is thought to challenge many aspects of proteostasis. The non-dividing, long-lived neurons are particularly vulnerable to stress, and, not surprisingly, even normal aging is highly associated with a decline in brain function in humans, as well as in other animals. Macroautophagy is a fundamental arm of the proteostasis network, safeguarding proper protein turnover during different cellular states and against diverse cellular stressors. An intricate interplay between macroautophagy and aging is beginning to unravel, with the emergence of new tools, including those for monitoring autophagy in cultured neurons and in the nervous system of different organisms in vivo. Here, we review recent findings on the impact of aging on neuronal integrity and on neuronal macroautophagy, as they emerge from studies in invertebrate and mammalian models

Cell biology in neuroscience: Death of developing neurons: new insights and implications for connectivity.

The concept that target tissues determine the survival of neurons has inspired much of the thinking on neuronal development in vertebrates, not least because it is supported by decades of research on nerve growth factor (NGF) in the peripheral nervous system (PNS). Recent discoveries now help to understand why only some developing neurons selectively depend on NGF. They also indicate that the survival of most neurons in the central nervous system (CNS) is not simply regulated by single growth factors like in the PNS. Additionally, components of the cell death machinery have begun to be recognized as regulators of selective axonal degeneration and synaptic function, thus playing a critical role in wiring up the nervous system

p107 regulates neural precursor cells in the mammalian brain

Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107−/− brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107−/− neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain

UniProt-Related Documents (UniReD): assisting wet lab biologists in their quest on finding novel counterparts in a protein network.

The in-depth study of protein-protein interactions (PPIs) is of key importance for understanding how cells operate. Therefore, in the past few years, many experimental as well as computational approaches have been developed for the identification and discovery of such interactions. Here, we present UniReD, a user-friendly, computational prediction tool which analyses biomedical literature in order to extract known protein associations and suggest undocumented ones. As a proof of concept, we demonstrate its usefulness by experimentally validating six predicted interactions and by benchmarking it against public databases of experimentally validated PPIs succeeding a high coverage. We believe that UniReD can become an important and intuitive resource for experimental biologists in their quest for finding novel associations within a protein network and a useful tool to complement experimental approaches (e.g. mass spectrometry) by producing sorted lists of candidate proteins for further experimental validation. UniReD is available at http://bioinformatics.med.uoc.gr/unired/

The Effect of Antiseptics on Adipose-Derived Stem Cells

Background: Although chemical antiseptics are the most basic measure to control wound infection and frequently come into contact with subcutaneous adipose tissue, no studies have evaluated their toxicity on adipose tissue and its cell fractions. In the present study, the effects of five different antiseptics on adipose-derived stem cells were evaluated. Methods: Human adipose-derived stem cells were harvested from healthy donors. Adipose-derived stem cell viability was measured after treatment with different concentrations of antiseptics over 5 days. Furthermore, the effect on the proliferation, adipogenic differentiation, and apoptosis/necrosis of adipose-derived stem cells was analyzed. Finally, the mRNA expression of the stem cell markers CD29, CD34, CD73, CD90, and CD105 was detected. Results: Octenisept and Betaisodona significantly reduced cell proliferation and differentiation and led to considerable adipose-derived stem cell necrosis. Octenisept decreased stem cell viability at the lowest concentrations tested, and all stem cell markers were down-regulated by Octeniseptr and Betaisodona. Lavasept and Prontosan both led to reduced stem cell viability, proliferation, and differentiation, and increased apoptosis/necrosis, although the effects were less pronounced compared with Octenisept and Betaisodona. Adipose-derived stem cells survived treatment with mafenide acetate even at high concentrations, and mafenide acetate showed minimal negative effects on their proliferation, adipogenic differentiation, cell death, and stem cell marker expression. Conclusions: Mafenide acetate may be regarded as a feasible antiseptic for the treatment of wounds with exposed adipose tissue because of its low adipose-derived stem cell toxicity. Lavasept and Prontosan are possible alternatives to mafenide acetate. Octenisept and Betaisodona, by contrast, may be used only in highly diluted solutions

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death