br Materials and methods br Results

Materials and methods
Results
Discussion Several in vivo and in vitro studies indicate that OPCs have substantially different properties according to their chronological age. For example, human fetal OPCs from different gestational stages exhibit differences in the myelination profile (Cui et al., 2012), such as human OPCs derived from fetal URMC-099 during the period of maximum oligoneogenesis or from adult subcortical white matter (Windrem et al., 2004). However, the mechanisms behind the different age-related OPC biological properties are not known. The aim of this study was to investigate if PARP is involved in determining the age-related OPCs properties. Thus, we compared OPCs derived from NSCs obtained from fetal and adult brains. This cell system was preferred to primary and purified OPCs to better mimic developmental biology of OPC, including lineage specification, proliferation/survival and differentiation. PARP is a family of enzymes comprising 17 homologues involved in multiple cell functions (Jubin et al., 2016a). Apart from the role in DNA repair, PARP-1 and 2 participate in cell homeostasis maintenance (Bai, 2015), intracellular transports (Abd Elmageed et al., 2012), cell cycle (Madison et al., 2011) inflammation and immunity (Bai and Virág, 2012) and gene expression regulation (Bock et al., 2015) through >100 substrates (Hottiger, 2015). Moreover, PARPs are critically involved in inflammation and in the tissue damage caused by ischemia/reperfusion conditions (Li et al., 2015). We first showed that PARP mRNA expression and PARP activity are much higher in fetal- than in adult-derived OPCs. Due to the role of PARPs in DNA repair and apoptosis induction (Heeres and Hergenrother, 2007), the reduction in PARP mRNA expression level and activity observed in adult compared to fetal-derived OPC might reflect the age-related decrease of DNA repair capability (Bürkle et al., 2015). In fact, PARP activity also varies according to the age in different brain regions (Strosznajder et al., 2000), and PARylation is developmentally regulated (Jubin et al., 2016b). We then showed that PARP inhibition produces substantially different effects in OPCs derived from fetal and adult brain. In particular: (i) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (ii) PARP inhibition reduces cell number in proportion to the inhibitory potency of the compounds; (iii) the PARP inhibition effect in fetal OPCs is a slow process (iv) PARP inhibition impairs OPC maturation into myelinating OL in fetal, but not in adult OPCs, according to the inhibitory potency of the compounds. A primary role of PARP in the differentiation process of different cell types was recently recognized, possibly though chromatin plasticity and epigenetic regulation (Li et al., 2015). In embryonic stem cells, PARP interacts with the Wnt pathway, Sonic Hedgehog and Pax6 signalling (Hemberger et al., 2003; Yoo et al., 2011). PARP-1 has also a role in neuroectoderm differentiation (Yoo et al., 2011) and favours the transit of NSCs toward a glial fate (Plane et al., 2012). Here we confirmed and further extend this latter observation, showing that PARP inhibition reduced proliferation in fetal-derived neurospheres and oligospheres (see supplemental material), and reduces OPCs differentiation into mature OLs. Notably, key transcription factors involved in OPC maturation, i.e. retinoic acid receptors, thyroid hormone receptors and their hetero-dimerization, such as PDGF signalling, a critical pathway for OPC biology, are targets of PARP regulation (Pavri et al., 2005; Allen, 2008). Overall, these results suggest that a different PARP signalling in fetal and adult OPCs might be part in the biological properties of OPCs at different chronological ages. These results have also therapeutic implications. In fact, PARP inhibition has even been proposed as a pharmacological strategy in a number of acquired inflammation/demyelinating disorders in which OPCs play a key pathogenic role, occurring both in perinatal/neonatal (e.g. neonatal hypoxia/ischemia encephalopathy) and adult age (e.g. spinal cord injury and multiple sclerosis; Komjáti et al., 2005; Moroni, 2008; Cavone and Chiarugi, 2012). Contradictory results have been obtained in mice models of inflammatory/demyelinating diseases (Selvaraj et al., 2009; Veto et al., 2010; Casaccia, 2011; Kamboj et al., 2013). PARP-2 deletion in conventional transgenic mice results in a protection from experimental allergic encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis (Kamboj et al., 2013). Conversely, knock-out of PARP-1 gene leads to an exacerbated EAE and an increase in the mortality rate (Selvaraj et al., 2009). Pharmacological PARP inhibition in EAE mice has a protective effect, preventing OL death and attenuating inflammation (Veto et al., 2010), although this effect is attributed to the reduction of CNS inflammation and immunomodulation (Scott et al., 2004).