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  • Netrin G NTNG part of the netrin DCC neogenin pathway

    2018-10-22

    Netrin-G1 (NTNG1; +2.2), part of the netrin/DCC/neogenin pathway, not only is an attractive guidance molecule for enteric neurons (Jiang et al., 2003), but also is involved in the outgrowth of thalamocortical neurons (Lin et al., 2003). Mutations in netrin-G1 are uncommon causes of the Rett syndrome, a disease where children loose hand skills and speech and also show distorted gut motility resulting in constipation or gastrointestinal reflux (Archer et al., 2006; Prior et al., 2010). Up- and downregulations of semaphorins (Sema3a, Sema3d, +1.6 and −1.6) or slit homolog 1 protein (Slit-1, −1.6) are also indicators for regulation of neurite and axon outgrowth (Shelly et al., 2011; Wolman et al., 2004; Dugan et al., 2011; Wong et al., 2012). In the mouse ENS, semaphorin 3A has been shown to regulate the entry of enteric neural precursor cells and endothelin receptor into the hindgut (Anderson et al., 2007). It is noteworthy that guidance molecules in the gut are expressed both in mesenchymal intestinal cells and enteric neural cells (Metzger et al., 2007b; Ratcliffe et al., 2011; Leibl et al., 1999). Since enterospheres consist of various mesenchymal and neural cell types (Kruger et al., 2002) we were able to detect the expression of different kinds of guidance molecules in our in vitro model.
    Competing interests
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    Acknowledgments
    Introduction AD is the most common age-related neurodegenerative disease estimated to affect approximately 30million people worldwide (Holtzman et al., 2011). Most prevalent symptoms are confusion and memory loss caused by synaptic dysfunction and neuronal death. One of the primary neuronal populations affected are the bfCNs, which are partly responsible for these cognitive deficits (Holtzman et al., 2011; Everitt and Robbins, 1997). The progress of AD research has been inhibited by lack of accurate models that recapitulate the complex facets of AD (Han et al., 2011). Recent advances in hPSC technology have made it possible to produce regionally specified neuronal populations affected by various neurodegenerative conditions, providing a novel source of human neurons for in vitro disease modeling (Nat and Dechant, 2011; Liu, 2011). It is essential that these neurons are phenotypically accurate and functional, and that they can also be used to model the embryonic differentiation of these populations, which is applicable to their regenerative potential (Liu, 2011). Previous studies have demonstrated the generation of cholinergic neurons, with a potential basal forebrain phenotype (Nilbratt et al., 2010; Bissonnette et al., 2011). However, high levels of specific extrinsic factors were used to direct differentiation, of which their exact role remains unclear. Our aim was to provide a method of generating high numbers of bfCNs in keeping with a requirement for a developmental model; a reductionist approach, where the cells establish their own developmental cues, in parallel to the developing embryo. Our reasoning for the advantages of this are two-fold; firstly we believe this approach provides a superior and more accurate developmental model to study the innate acquisition of basal forebrain cholinergic fate; furthermore by using intrinsic cues from development we would suggest that the resulting neuronal progeny would be more similar to those in the developing brain. We present evidence that a method of EB-based non-adherent differentiation (NAdD) is sufficient for the induction of basal forebrain fate. In the embryo, bfCNs differentiate in the ventral telencephalon, which will go on to form part of the basal forebrain (Marin et al., 2000). Hedgehog signaling is the master controller of dorso-ventral patterning, inducing ventral fate along the entire length of the developing neural tube (Briscoe and Ericson, 1999). We show that NAdD results in the production of the secreted hedgehog ligand SHH, which as in the embryonic ventral telencephalon, results in expression of the specific transcription factors NKX2.1 and LHX8 (Marin et al., 2000; Ericson et al., 1995a; Shimamura et al., 1995; Pera and Kessel, 1997; Gunhaga et al., 2000; Sussel et al., 1999; Flandin et al., 2010; Zhao et al., 2003; Fragkouli et al., 2005). Optimization of the NAdD protocol generated an expandable population of NKX2.1+/LHX8+ NSCs, which retained this phenotype through long-term expansion. Upon terminal differentiation the NSCs generated TUJ1+/ChAT+ cholinergic neurons, demonstrating characteristics of bfCNs present in the adult human brain. Briefly they expressed high levels of p75NTR protein, as well as cholinergic receptor subunit genes, essential for their function and upon transplantation the NSCs were also able to differentiate into cholinergic neurons in the adult rat brain. Furthermore, the hPSC-derived bfCNs were electrically active, released acetylcholine, and generated neuronal action potential firing as well as spontaneous activity. Therefore our work provides a model of the human bfCN population, which meets the criteria required for a multipurpose model of the basal forebrain cholinergic system, both developmentally and also in a mature functional context.