Here using several murine and human cellular

Here using several murine and human cellular models, we present compelling evidence for ASYN expression during oligodendrocyte lineage cell development. Moreover, we identified both ASYN protein and transcripts in purified PDGFRA/CD140a+ oligodendrocyte progenitors and oligodendrocyte lineage SOX10+ nuclei, isolated by fluorescence-activated cell sorting (FACS) from the rodent wild-type BMN-673 Supplier and human healthy and diseased brains, respectively, therefore substantiating a functional role for ASYN in oligodendrocytes.
Results
Discussion The absence of SNCA transcripts in oligodendrocytes in the adult brain of healthy and MSA patients has been reported repeatedly (Iwai et al., 1995; Miller et al., 2005; Solano et al., 2000), but recent studies have challenged these results. Because MSA has been identified as a non-genetic disorder, it is difficult to propose an explanation for the origin of ASYN in GCIs present in oligodendrocytes under pathological condition. This has led to the emergence of a hypothesis advocating a mechanism of cell-to-cell transfer of exogenous ASYN to oligodendrocytes, similar to that experimentally established from neuron-to-neuron in PD models (Hansen et al., 2011). However, the recent use of more advanced techniques, such as laser-capture micro-dissection, has allowed for the isolation of adult white matter glia from human postmortem tissue, resulting in the detection of SNCA transcripts from MSA, idiopathic PD, and control cases (Asi et al., 2014). We clearly identified ASYN protein by ICC and western blotting and we detected transcripts by quantitative real-time PCR in oligodendrocyte lineage cells from rodent and human origins (summarized in Figure 4J). We could not detect ASYN expression in astrocytes and microglia, which are cell types known to take up exogenous ASYN (Boza-Serrano et al., 2014; Lee et al., 2010). Moreover, the few neurons present in cultures combined with the multitude of media changes applied during the culture period, and the discovery of Snca/SNCA transcripts in freshly isolated oligodendrocytes and ASYN protein in homogenous cultures of freshly isolated, maturing PDGFRA/CD140a+ oligodendrocyte progenitors, together allow us to rule out the possibility that healthy or dying neurons, even contaminating neurons, may have released large quantities of ASYN, which would accumulate, not being sufficiently degraded, in the oligodendrocytes only. Moreover, we showed relocation of ASYN during oligodendrocyte differentiation from the processes to the perinuclear space, where the protein appears as a single dot-like structure suggesting it is finally degraded. We identified SNCA transcripts in human SOX10+ oligodendrocyte lineage nuclei samples isolated from healthy and MSA patients (Figure 4). This result is in line with recent observations (Asi et al., 2014). Whether ASYN expression identified in these two studies is reminiscent of a basal expression in quiescent OPCs, and the difference although not significant identified in oligodendrocytes from MSA and control samples is the result of a greater oligodendrogenesis probably triggered by neuronal injury (Ahmed et al., 2013) remain to be determined. We found a decrease of ASYN associated with oligodendrocyte maturation characterized by complex cellular processes and expression of the mature marker MBP. Interestingly, overexpression of ASYN in OPCs delays their maturation into MBP+ oligodendrocytes (Ettle et al., 2014). Additionally, we found that ASYN is decreased in pre-myelinating oligodendrocytes, regardless of the genetic background of the iPSC (diseased or healthy). This observation, although made in vitro, may allow us to rule out that the origin of ASYN in GCIs in MSA, and glial inclusions in PD, may result from the accumulation of a lifetime production of ASYN throughout glial lineages. Successful generation of patient-specific iPSCs, and their subsequent differentiation into brain cell types, has opened up unlimited access of patient brain cells to a growing number of investigators interested in studying neurodegenerative diseases. Here we have pioneered the generation of oligodendrocytes from MSA and familial PD iPSC lines. Our protocol for generating oligodendrocytes from human iPSCs, therefore, adds on to those recently developed for studying demyelination diseases (Douvaras et al., 2014; Numasawa-Kuroiwa et al., 2014; Piao et al., 2015; Stacpoole et al., 2013; Wang et al., 2013). Importantly, our iPSC models provide a greater number of lines available for studying synucleinopathies.