Oligodendrocyte precursor cells (OPCs) are the main actors involved in developmental myelination and adult myelination dynamic, including remyelination after myelin injury. The recapitualtion hypothesis states that remyelination in adulthood is a complete recapitulation of developmental myelination. However, the different nature of foetal and adult OPCs could reflect a different myelination mechanism and different response to demyelinating insults. The main objective of the present study is the

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... investigation of foetal and adult OPCs vulnerability to the two main components of the demyelinating diseases: inflammation and hypoxia/ischemia (HI). In order to develop the main objective, robust and efficient in vitro models and readouts were set up. In particular, a cell-based high content screening approach was used as the elective technique in order to obtain robust and reproducible data. Moreover, since the differential response of foetal and adult OPCs could come from a different physiological differentiation process, driven by thyroid hormone (TH) and exerted by nuclear receptors (NRs) activity, we first focused on the role of NRs and co-regulator in foetal and adult OPCs differentiation. We found a different expression of NRs in early precursors, a different response to T3-mediated differentiation, the selective importance of PARP activity for foetal OPCs and the fundamental role of RXRγ in both systems. Differences in physiological differentiation can reflect different responses to noxious stimuli. Thus, we exposed OPCs to inflammatory cytokines mix, in order to mimic the inflammation environment, or to oxygen-glucose deprivation (OGD), in order to mimic HI. OGD was first characterized in primary neuronal cultures and tested for its reliability in disease model studies and pharmacological analysis. Foetal and adult OPCs show both a cytokines-mediated differentiation block, reflecting what the in vivo pathological condition. We thus tested a pharmacological tool in adult cultures, in order to study the mechanism. In particular, we found that the differentiation block was linked to an increase of deiodinase 3 (D3), the enzyme inactivating T3, the active form of TH and, blocking its activity, it was possible to restore the OPC differentiation process. On the other hand, only foetal OPCs are sensitive to OGD, showing OPCs/OLs-specific cell death. Since we found an increase in D3 expression after OGD in foetal OPCs, further studies will elucidate the mechanism underlying the foetal-selective OPCs cell death, analysing the role of D3 in the process and glutamate-mediated excitotxicity, the major triggerer of HIinduced cell death. The better understanding of OPCs biology and mechanisms underlying physiological and pathological conditions, bound to information about differences in foetal and adult OPCs, will open the way to new therapies development, based on differential strategies for demyelinating diseases affecting foetal or adult life. Is remyelination a recapitulation of developmental myelination? Myelination and remyelination share a common objective: wrap non-myelinating axons, to create the correct cellular and molecular structure for an efficient saltatory axonal conduction. The recapitulation hypothesis of myelin regeneration holds that mechanisms that underlie remyelination after injury are essentially a rerunning of a developmental myelination program . For example, two of the best studied pathways, identified as crucial players in regulating developmental myelination, have been subsequently found to be also limiting factors for remyelination: Notch signaling pathway and the leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1 (LINGO-1), two strong inhibitors of OPCs differentiation and myelination. Both factors are expressed during development and contribute to the control of differentiation timing and, in different demyelinating conditions, they are highly expressed, contributing to the pathogenesis (Bhatt et al., 2014). However, in spite of the evidences remarking the recapitulation hypothesis, morphological differences between the two processes lead to the view that these two mechanisms are differentially regulated (Miron, 2011). The OPCs generated by the three different waves during development, produce the adult OPCs pool. Thus, these cells are different spatiotemporally defined OPCs classes, differing from their perinatal forebears for antigenic markers, growth factor responsiveness, basal motility rates and cell cycle (Fancy et al., 2011). Moreover, compared to foetal OPCs, adult progenitors show a longer cell cycle time and a slower rate of migration. Little is known about transcription factors roles during remyelination, but Olig1 and Olig2 seem to play different roles in the two processes. According to some authors, Olig1 is essential for myelination but not for remyelination (Bradl and Lassmann, 2008), but other authors believe that is essential also for remyelination . It is well known, instead, that Olig2 is fundamental for remyelination (Bradl and Lassmann, 2008). Abstract Nuclear receptors (NRs) play key roles in the oligodendrocytes (OLs) differentiation, from NSCs self-renewal and lineage specification, to OLs maturation. Foetal and adult oligodendrocyte precursor cells (OPCs) share the same objective: to wrap and functionally myelinate axons, in a process mediated by thyroid hormone (TH), and acting throughout NRssignals. In particular, thyroid hormone receptors (TRs) are the main NRs involved in the process, acting as homodimer or heterodimer with others NRs, expecially RXRs. Here we showed that neural stem cells (NSCs), the progenitors of OPCs, isolated from foetal and adult brain, differentially express two fundamental NRs involved in the differentiation process: TRβ and RXRγ. We showed that both systems need TH to differentiate in mature OLs. However, even if adult cells show a more mature profile in early stages, they are less responsive to TH differentiating signal. Moreover, we demonstrated that RXRγ is fundamental in both systems to achieve differentiation, showing a severe reduction in mature/myelinating OLs in cultures isolated from RXRγ-/-mice. However, RXRγ-/-cultures show the same gene expression regulation in response to T3-differentiation induction, compares to RXRγ+/+ cultures, suggesting that it is not directly involved in the molecular machinery of the maturation process. RXRγ-/-cultures show an higher percentage of replicating OPCs and do not show reduction of proliferation in response to T3, indicating a role of RXRγ in the control of cell cycle exit. Differences and similarity in T3-mediated OPCs differentiation will open new strategies in understanding their biology and developing new therapies in demyelinating disease, differentially affecting foetal or adult OPCs.