Loss of protein-tyrosine phosphatase α (PTPα) increases proliferation and delays maturation of oligodendrocyte progenitor cells

Tightly controlled termination of proliferation determines when oligodendrocyte progenitor cells (OPCs) can initiate differentiation and mature into myelin-forming cells. Protein-tyrosine phosphatase α (PTPα) promotes OPC differentiation, but its role in proliferation is unknown. Here we report that loss of PTPα enhanced in vitro proliferation and survival and decreased cell cycle exit and growth factor dependence of OPCs but not neural stem/progenitor cells. PTPα(-/-) mice have more oligodendrocyte lineage cells in embryonic forebrain and delayed OPC maturation. On the molecular level, PTPα-deficient mouse OPCs and rat CG4 cells have decreased Fyn and increased Ras, Cdc42, Rac1, and Rho activities, and reduced expression of the Cdk inhibitor p27Kip1. Moreover, Fyn was required to suppress Ras and Rho and for p27Kip1 accumulation, and Rho inhibition in PTPα-deficient cells restored expression of p27Kip1. We propose that PTPα-Fyn signaling negatively regulates OPC proliferation by down-regulating Ras and Rho, leading to p27Kip1 accumulation and cell cycle exit. Thus, PTPα acts in OPCs to limit self-renewal and facilitate differentiation.     

Telomerase and oligodendrocyte differentiation.

Myelin in the mammalian central nervous system (CNS) is produced by oligodendrocytes, most of which arise from oligodendrocyte precursor cells (OPCs) during late embryonic and early postnatal development. Both external and internal cues have been implicated in regulating OPC exit from the cell cycle and differentiation into oligodendrocytes. In this study, we demonstrate that differentiation of cultured OPCs into mature oligodendrocytes is associated with lower levels of activity of telomerase, the ribonucleoprotein that synthesizes telomeric DNA at the ends of chromosomes. Differentiation is also associated with lower levels of mRNA encoding the catalytic subunit of telomerase (TERT), whereas no difference is seen in the expression of its telomeric template RNA component (TR). These data suggest a possible role for telomerase during normal growth and differentiation of oligodendrocytes that may be relevant to the mechanism of myelination in the CNS.     

Telomerase and oligodendrocyte differentiation

Myelin in the mammalian central nervous system (CNS) is produced by oligodendrocytes, most of which arise from oligodendrocyte precursor cells (OPCs) during late embryonic and early postnatal development. Both external and internal cues have been implicated in regulating OPC exit from the cell cycle and differentiation into oligodendrocytes. In this study, we demonstrate that differentiation of cultured OPCs into mature oligodendrocytes is associated with lower levels of activity of telomerase, the ribonucleoprotein that synthesizes telomeric DNA at the ends of chromosomes. Differentiation is also associated with lower levels of mRNA encoding the catalytic subunit of telomerase (TERT), whereas no difference is seen in the expression of its telomeric template RNA component (TR). These data suggest a possible role for telomerase during normal growth and differentiation of oligodendrocytes that may be relevant to the mechanism of myelination in the CNS. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 224–234, 2001     

Oscillatory expression of Ascl1 in oligodendrogenesis

The proneural gene Ascl1 promotes formation of both neurons and oligodendrocytes from neural stem cells (NSCs), but it remains to be analyzed how its different functions are coordinated. It was previously shown that Ascl1 enhances proliferation of NSCs when its expression oscillates but induces differentiation into transit-amplifying precursor cells and neurons when its expression is up-regulated and sustained. By time-lapse imaging and immunohistological analyses, we found that Ascl1 expression oscillated in proliferating oligodendrocyte precursor cells (OPCs) at lower levels than in transit-amplifying precursor cells and was repressed when OPCs differentiated into mature oligodendrocytes. Induction of sustained overexpression of Ascl1 reduced oligodendrocyte differentiation and promoted neuronal differentiation. These results suggest that oscillatory expression of Ascl1 plays an important role in proliferating OPCs during oligodendrocyte formation.     

Impact of Simulated Microgravity on Oligodendrocyte Development: Implications for Central Nervous System Repair

We have recently established a culture system to study the impact of simulated microgravity on oligodendrocyte progenitor cells (OPCs) development. We subjected mouse and human OPCs to a short exposure of simulated microgravity produced by a 3D-Clinostat robot. Our results demonstrate that rodent and human OPCs display enhanced and sustained proliferation when exposed to simulated microgravity as assessed by several parameters, including a decrease in the cell cycle time. Additionally, OPC migration was examined in vitro using time-lapse imaging of cultured OPCs. Our results indicated that OPCs migrate to a greater extent after stimulated microgravity than in normal conditions, and this enhanced motility was associated with OPC morphological changes. The lack of normal gravity resulted in a significant increase in the migration speed of mouse and human OPCs and we found that the average leading process in migrating bipolar OPCs was significantly longer in microgravity treated cells than in controls, demonstrating that during OPC migration the lack of gravity promotes leading process extension, an essential step in the process of OPC migration. Finally, we tested the effect of simulated microgravity on OPC differentiation. Our data showed that the expression of mature oligodendrocyte markers was significantly delayed in microgravity treated OPCs. Under conditions where OPCs were allowed to progress in the lineage, simulated microgravity decreased the proportion of cells that expressed mature markers, such as CC1 and MBP, with a concomitant increased number of cells that retained immature oligodendrocyte markers such as Sox2 and NG2. Development of methodologies aimed at enhancing the number of OPCs and their ability to progress on the oligodendrocyte lineage is of great value for treatment of demyelinating disorders. To our knowledge, this is the first report on the gravitational modulation of oligodendrocyte intrinsic plasticity to increase their progenies.     

MiRNA‐145‐5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR‐145‐5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR‐145‐5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG⁺ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR‐145‐5p knockdown OPCs. Further, knockdown of miR‐145‐5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL‐specific genes targeted by miR‐145‐5p that exhibited upregulation with miR‐145‐5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR‐145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR‐145‐5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR‐145‐5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR‐145‐5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as‐yet‐unidentified mechanisms. These findings clarify the need for differential regulation of miR‐145‐5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR‐145‐5p is dysregulated.     

Interactions between oligodendrocyte precursors control the onset of CNS myelination

The formation of CNS myelin is dependent on the differentiation of oligodendrocyte precursor cells (OPCs) and oligodendrocyte maturation. How the initiation of myelination is regulated is unclear, but it is likely to depend on the development of competence by oligodendrocytes and receptivity by target axons. Here we identify an additional level of control of oligodendrocyte maturation mediated by interactions between the different cellular components of the oligodendrocyte lineage. During development oligodendrocyte precursors mature through a series of stages defined by labeling with monoclonal antibodies A2B5 and O4. Newly differentiated oligodendrocytes begin to express galactocerebroside recognized by O1 antibodies and subsequently mature to myelin basic protein (MBP)-positive cells prior to formation of compact myelin. Using an in vitro brain slice culture system that supports robust myelination, the consequences of ablating cells at different stages of the oligodendrocyte lineage on myelination have been assayed. Elimination of all OPC lineage cells through A2B5+, O4+, and O1+ complement-mediated cell lysis resulted in a delay in development of MBP cells and myelination. Selective elimination of early OPCs (A2B5+) also unexpectedly resulted in delayed MBP expression compared to controls suggesting that early OPCs contribute to the timing of myelination onset. By contrast, elimination of differentiated (O1+) immature oligodendrocytes permanently inhibited the appearance of MBP+ cells suggesting that oligodendrocytes are critical to facilitate the maturation of OPCs. These data illuminate that the presence of intra-lineage feed-forward and feedback cues are important for timely myelination by oligodendrocytes.     

Dynamic Changes of CD44 Expression from Progenitors to Subpopulations of Astrocytes and Neurons in Developing Cerebellum

We previously reported that CD44-positive cells were candidates for astrocyte precursor cells in the developing cerebellum, because cells expressing high levels of CD44 selected by fluorescence-activated cell sorting (FACS) gave rise only to astrocytes in vitro. However, whether CD44 is a specific cell marker for cerebellar astrocyte precursor cells in vivo is unknown. In this study, we used immunohistochemistry, in situ hybridization, and FACS to analyze the spatial and temporal expression of CD44 and characterize the CD44-positive cells in the mouse cerebellum during development. CD44 expression was observed not only in astrocyte precursor cells but also in neural stem cells and oligodendrocyte precursor cells (OPCs) at early postnatal stages. CD44 expression in OPCs was shut off during oligodendrocyte differentiation. Interestingly, during development, CD44 expression was limited specifically to Bergmann glia and fibrous astrocytes among three types of astrocytes in cerebellum, and expression in astrocytes was shut off during postnatal development. CD44 expression was also detected in developing Purkinje and granule neurons but was limited to granule neurons in the adult cerebellum. Thus, at early developmental stages of the cerebellum, CD44 was widely expressed in several types of precursor cells, and over the course of development, the expression of CD44 became restricted to granule neurons in the adult.     

Normal timing of oligodendrocyte development depends on thyroid hormone receptor alpha 1 (TRalpha1)

The timing of oligodendrocyte development is regulated by thyroid hormone (TH) in vitro and in vivo, but it is still uncertain which TH receptors mediate this regulation. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Here, we provide direct evidence for the involvement of the TRalpha1 receptor isoform in vivo, by showing that the number of oligodendrocytes in the postnatal day 7 (P7) and P14 optic nerve of TRalpha1-/- mice is decreased compared with normal. We demonstrate that TRalpha1 mediates the normal differentiation-promoting effect of TH on oligodendrocyte precursor cells (OPCs): unlike wild-type OPCs, postnatal TRalpha1-/- OPCs fail to stop dividing and differentiate in response to TH in culture. We also show that overexpression of TRalpha1 accelerates oligodendrocyte differentiation in culture, suggesting that the level of TRalpha1 expression is normally limiting for TH-dependent OPC differentiation. Finally, we provide evidence that the inhibitory isoforms of TRalpha are unlikely to play a part in the timing of OPC differentiation.     

Differential proliferation rhythm of neural progenitor and oligodendrocyte precursor cells in the young adult hippocampus

Oligodendrocyte precursor cells (OPCs) are a unique type of glial cells that function as oligodendrocyte progenitors while constantly proliferating in the normal condition from rodents to humans. However, the functional roles they play in the adult brain are largely unknown. In this study, we focus on the manner of OPC proliferation in the hippocampus of the young adult mice. Here we report that there are oscillatory dynamics in OPC proliferation that differ from neurogenesis in the subgranular zone (SGZ); the former showed S-phase and M-phase peaks in the resting and active periods, respectively, while the latter only exhibited M-phase peak in the active period. There is coincidence between different modes of proliferation and expression of cyclin proteins that are crucial for cell cycle; cyclin D1 is expressed in OPCs, while cyclin D2 is observed in neural stem cells. Similar to neurogenesis, the proliferation of hippocampal OPCs was enhanced by voluntary exercise that leads to an increase in neuronal activity in the hippocampus. These data suggest an intriguing control of OPC proliferation in the hippocampus.     

Role of thyroid hormone receptors in timing oligodendrocyte differentiation.

The timing of oligodendrocyte differentiation is thought to depend on both intracellular mechanisms and extracellular signals. Thyroid hormone (TH) helps control this timing both in vitro and in vivo, but it is still uncertain how it does so. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Previous studies suggested that TRbeta receptors may mediate the effect of TH on oligodendrocyte precursor cells (OPCs). Consistent with this possibility, we show here that overexpression of TRbeta1 promotes precocious oligodendrocyte differentiation, whereas expression of two dominant-negative forms of TRbeta1 greatly delays differentiation. Surprisingly, however, we find that postnatal TRbeta-/- mice have a normal number of oligodendrocytes in their optic nerves and that TRbeta-/- OPCs stop dividing and differentiate normally in response to TH in vitro. Moreover, we find that OPCs do not express TRbeta1 or TRbeta2 mRNAs, whereas they do express TRalpha1 and TRalpha2 mRNAs. These findings suggest that alpha receptors mediate the effect of TH on the timing of oligodendrocyte differentiation. We also show that TRalpha2 mRNA, which encodes a dominant-negative form of TRalpha, decreases as OPCs proliferate in vitro and in vivo. This decrease may help control when oligodendrocyte precursors differentiate.     

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.     

Delta-Notch signaling regulates oligodendrocyte specification

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analysing mutant zebrafish embryos, we found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, we also learned that constitutive Notch activity could promote formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. Our data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate.     

Role of the cellular prion protein in oligodendrocyte precursor cell proliferation and differentiation in the developing and adult mouse CNS

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrP(c)) to this process remains unclear. PrP(c) is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrP(c) influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrP(c) proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrP(c) knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells.     

The RcsCB His-Asp phosphorelay system is essential to overcome chlorpromazine-induced stress in Escherichia coli

The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.     

The selective RNA-binding protein quaking I (QKI) is necessary and sufficient for promoting oligodendroglia differentiation

Quaking I (QKI) is a selective RNA-binding protein essential for myelination of the central nervous system. Three QKI isoforms with distinct C termini and subcellular localization, namely QKI-5, QKI-6, and QKI-7, are expressed in oligodendroglia progenitor cells (OPCs) prior to the initiation of myelin formation and implicated in promoting oligodendrocyte lineage development. However, the functional requirement for each QKI isoform and the mechanisms by which QKI isoforms govern OPC development still remain elusive. We report here that exogenous expression of each QKI isoform is sufficient to enhance differentiation of OPCs with different efficiency, which is abolished by a point mutation that abrogates the RNA binding activity of QKI. Reciprocally, small interfering RNA-mediated QKI knockdown blocks OPC differentiation, which can be partly rescued by QKI-5 and QKI-6 but not by QKI-7, indicating the differential requirement of QKI isoform function in advancing OPC differentiation. Furthermore, we found that abrogation of OPC differentiation, as a result of QKI deficiency, is not due to altered proliferation capacity or cell cycle progression. These results indicate that QKI isoforms are necessary and sufficient for promoting OPC development, which must involve direct influence of QKI on differentiation/maturation of OPCs independent of cell cycle exit, likely via regulating the expression of the target mRNAs of QKI that support OPC differentiation.     

Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain

Both late-gestation and adult human forebrain contain large numbers of oligodendrocyte progenitor cells (OPCs). These cells may be identified by their A2B5(+)PSA-NCAM(-) phenotype (positive for the early oligodendrocyte marker A2B5 and negative for the polysialylated neural cell adhesion molecule). We used dual-color fluorescence-activated cell sorting (FACS) to extract OPCs from 21- to 23-week-old fetal human forebrain, and A2B5 selection to extract these cells from adult white matter. When xenografted to the forebrains of newborn shiverer mice, fetal OPCs dispersed throughout the white matter and developed into oligodendrocytes and astrocytes. By 12 weeks, the host brains showed extensive myelin production, compaction and axonal myelination. Isolates of OPCs derived from adult human white matter also myelinated shiverer mouse brain, but much more rapidly than their fetal counterparts, achieving widespread and dense myelin basic protein (MBP) expression by 4 weeks after grafting. Adult OPCs generated oligodendrocytes more efficiently than fetal OPCs, and ensheathed more host axons per donor cell than fetal cells. Both fetal and adult OPC phenotypes mediated the extensive and robust myelination of congenitally dysmyelinated host brain, although their differences suggested their use for different disease targets.