Heterogeneous fate choice of genetically modulated adult neural stem cells in gray and white matter of the central nervous system.

Apart from dedicated oligodendroglial progenitor cells, adult neural stem cells (aNSCs) can also give rise to new oligodendrocytes in the adult central nervous system (CNS). This process mainly confers myelinating glial cell replacement in pathological situations and can hence contribute to glial heterogeneity. Our previous studies demonstrated that the p57kip2 gene encodes an intrinsic regulator of glial fate acquisition and we here investigated to what degree its modulation can affect stem cell-dependent oligodendrogenesis in different CNS environments. We therefore transplanted p57kip2 knockdown aNSCs into white and gray matter (WM and GM) regions of the mouse brain, into uninjured spinal cords as well as in the vicinity of spinal cord injuries and evaluated integration and differentiation in vivo. Our experiments revealed that under healthy conditions intrinsic suppression of p57kip2 as well as WM localization promote differentiation toward myelinating oligodendrocytes at the expense of astrocyte generation. Moreover, p57kip2 knockdown conferred a strong benefit on cell survival augmenting net oligodendrocyte generation. In the vicinity of hemisectioned spinal cords, the gene knockdown led to a similar induction of oligodendroglial features; however, newly generated oligodendrocytes appeared to suffer more from the hostile environment. This study contributes to our understanding of mechanisms of adult oligodendrogenesis and glial heterogeneity and further reveals critical factors when considering aNSC mediated cell replacement in injury and disease.

The role of oligodendrocyte precursor cells expressing the GPR17 receptor in brain remodeling after stroke.

Following stroke-induced neuronal damage, quiescent oligodendrocyte precursors (OPCs) are activated to proliferate and later to differentiate to myelin-producing cells. GPR17, a receptor transiently expressed on early OPCs, has emerged as a target to implement stroke repair through stimulation of OPC maturation. However, being GPR17 completely downregulated in myelin-producing oligodendrocytes, its actual role in determining the final fate of OPCs after cerebral ischemia is still uncertain. Here, to univocally define the spatiotemporal changes and final fate of GPR17-expressing OPCs, we induced ischemia by middle cerebral artery occlusion (MCAo) in reporter GPR17iCreERT2:CAG-eGreen florescent protein (GFP) mice, in which, upon tamoxifen treatment, cells expressing GPR17 become green and traceable for their entire life. Starting from 3 days and up to 2 weeks after MCAo, GFP+ cells markedly accumulated in regions surrounding the ischemic lesion; several of them proliferated, as shown by co-labeling of the DNA synthesis marker 5-Bromo-2'-deoxyuridine (BrdU). Almost all GFP+/BrdU+ cells expressed the OPC early marker neural/glial antigen 2 (NG2), indicating that they were still precursors. Accumulation of GFP+ cells was also because of OPC recruitment from surrounding areas, as suggested in vivo by acquisition of typical features of migrating OPCs, shown in vitro in presence of the chemoattractant PDGF-AA and confirmed by transplantation of GFP+-OPCs in wild-type MCAo mice. Eight weeks after MCAo, only some of these precociously recruited cells had undergone maturation as shown by NG2 loss and acquisition of mature myelinating markers like GSTpi. A pool of recruited GFP+-OPCs was kept at a precursor stage to likely make it available for further insults. Thus, very early after ischemia, GFP+-OPCs proliferate and migrate toward the lesion; however, most of these cells remain undifferentiated, suggesting functional roles other than myelination.

GPR17 expressing NG2-Glia: Oligodendrocyte progenitors serving as a reserve pool after injury.

In the adult brain NG2-glia continuously generate mature, myelinating oligodendrocytes. To which extent the differentiation process is common to all NG2-glia and whether distinct pools are recruited for repair under physiological and pathological conditions still needs clarification. Here, we aimed at investigating the differentiation potential of adult NG2-glia that specifically express the G-protein coupled receptor 17 (GPR17), a membrane receptor that regulates the differentiation of these cells at postnatal stages. To this aim, we generated the first BAC transgenic GPR17-iCreER(T2) mouse line for fate mapping studies. In these mice, under physiological conditions, GPR17(+) cells--in contrast to GPR17(-) NG2-glia--did not differentiate within 3 months, a peculiarity that was overcome after cerebral damage induced by acute injury or ischemia. After these insults, GPR17(+) NG2-glia rapidly reacted to the damage and underwent maturation, suggesting that they represent a 'reserve pool' of adult progenitors maintained for repair purposes.

Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex.

The adult cerebral cortex lacks the capacity to replace degenerated neurons following traumatic injury. Conversion of nonneuronal cells into induced neurons has been proposed as an innovative strategy toward brain repair. Here, we show that retrovirus-mediated expression of the transcription factors Sox2 and Ascl1, but strikingly also Sox2 alone, can induce the conversion of genetically fate-mapped NG2 glia into induced doublecortin (DCX)(+) neurons in the adult mouse cerebral cortex following stab wound injury in vivo. In contrast, lentiviral expression of Sox2 in the unlesioned cortex failed to convert oligodendroglial and astroglial cells into DCX(+) cells. Neurons induced following injury mature morphologically and some acquire NeuN while losing DCX. Patch-clamp recording of slices containing Sox2- and/or Ascl1-transduced cells revealed that a substantial fraction of these cells receive synaptic inputs from neurons neighboring the injury site. Thus, NG2 glia represent a potential target for reprogramming strategies toward cortical repair.

Transplantation reveals regional differences in oligodendrocyte differentiation in the adult brain.

To examine the role of gray and white matter niches for oligodendrocyte differentiation, we used homo- and heterotopic transplantations into the adult mouse cerebral cortex. White matter-derived cells differentiated into mature oligodendrocytes in both niches with equal efficiency, whereas gray matter-derived cells did not. Thus, white matter promotes oligodendrocyte differentiation, and cells from this niche differentiate more easily, even in the less supportive gray matter environment.

The GPR17 receptor in NG2 expressing cells: focus on in vivo cell maturation and participation in acute trauma and chronic damage.

NG2-expressing cells comprise a population of cycling precursors that can exit the cell cycle and differentiate into mature oligodendrocytes. As a whole, they display heterogeneous properties and behaviors that remain unresolved at the molecular level, although partly interpretable as distinct maturation stages. To address this issue, we analyzed the expression of the GPR17 receptor, recently shown to decorate NG2-expressing cells and to operate as an early sensor of brain damage, in immature and adult oligodendrocyte progenitors in the intact brain and after injury. In both the early postnatal and adult cerebral cortex, distinct GPR17 protein localizations and expression levels define different stages of oligodendroglial maturation, ranging from the precursor phase to the premyelinating phenotype. As soon as cells exit mitosis, a fraction of NG2-expressing cells displays accumulation of GPR17 protein in the Golgi apparatus. GPR17 expression is subsequently upregulated and distributed to processes of cells that stop dividing, progressively lose NG2 positivity and assume premyelinating features. Absence of colabeling with mature markers or myelin proteins indicates that GPR17 is downregulated when cells complete their final maturation. BrdU-based fate-mapping demonstrated that a significant fraction of newly generated oligodendrocyte progenitors transiently upregulates GPR17 during maturation. Importantly, we also found that GPR17 does not participate to the early reaction of NG2-expressing cells to damage, while it is induced at postacute stages after injury. These findings identify GPR17 as a marker for progenitor progression within the oligodendroglial lineage and highlight its participation to postacute reactivity of NG2 cells in different injury paradigms.

Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier.

The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.