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

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

Neurotrophin receptors TrkA and TrkC cause neuronal death whereas TrkB does not

12 páginasNeurons of the peripheral nervous system have long been known to require survival factors to prevent their death during development. But why they selectively become dependent on secretory molecules has remained a mystery, as is the observation that in the central nervous system, most neurons do not show this dependency. Using engineered embryonic stem cells, we show here that the neurotrophin receptors TrkA and TrkC (tropomyosin receptor kinase A and C, also known as Ntrk1 and Ntrk3, respectively) instruct developing neurons to die, both in vitro and in vivo. By contrast, TrkB (also known as Ntrk2), a closely related receptor primarily expressed in the central nervous system, does not. These results indicate that TrkA and TrkC behave as dependence receptors, explaining why developing sympathetic and sensory neurons become trophic-factor-dependent for survival. We suggest that the expansion of the Trk gene family that accompanied the segregation of the peripheral from the central nervous system generated a novel mechanism of cell number controlH.L. is supported by an Emmy-Nother fellowship of the DFG.Peer reviewe