Qiao-Ling Cui

IGF-I-induced oligodendrocyte progenitor proliferation requires PI3K/Akt, MEK/ERK, and Src-like tyrosine kinases

Insulin‐like growth factor‐I (IGF‐I) is required for the growth of oligodendrocytes, although the underlying mechanisms are not fully understood. Our aim was to investigate the role of phosphatidylinositol 3‐kinase (PI3K), mitogen‐activated protein kinase kinase (MEK1), and Src family tyrosine kinases in IGF‐I‐stimulated proliferation of oligodendrocyte progenitors. IGF‐I treatment increased the proliferation of cultured oligodendrocyte progenitors as determined by measuring incorporation of [3H]‐thymidine and bromodeoxy‐uridine (BrdU). IGF‐I stimulated a transient phosphorylation of 3‐phosphoinositide‐dependent kinase‐1 (PDK1) and extracellular signal‐regulated kinases (ERK1/2) (targets of MEK1), as well as a rapid and sustained activation of Akt (a target of PI3K). Furthermore, inhibitors of PI3K (LY294002 and Wortmannin), MEK1 (PD98059 and U0126), and Src family tyrosine kinases (PP2) decreased IGF‐I‐induced proliferation, and blocked ERK1/2 activation. LY294002, Wortmannin and PP2 also blocked Akt activation. To further determine whether Akt is required for IGF‐I stimulated oligodendrocyte progenitor proliferation, cultures were infected with adenovirus vectors expressing dominant‐negative mutants of Akt or treated with pharmacological inhibitors of Akt. All treatments reduced IGF‐I‐induced oligodendrocyte progenitor proliferation. Our data indicate that stimulation of oligodendrocyte progenitor proliferation by IGF‐I requires Src‐like tyrosine kinases as well as the PI3K/Akt and MEK1/ERK signaling pathways.

Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways

The function of muscarinic acetylcholine receptors expressed in oligodendrocytes and in myelin has remained largely undetermined. Here we present evidence that incubation of oligodendrocyte progenitors, deprived of growth factor, with the acetylcholine analog carbachol significantly reduced cell death by apoptosis and blocked caspase-3 cleavage. This protective effect was reversed by atropine, a muscarinic acetylcholine receptor antagonist, as well as by specific inhibitors of intracellular signaling molecules, including phosphatidylinositol 3-kinase (Wortmannin and LY294002), Akt (Akt inhibitor III) and Src-like tyrosine kinases (PP2), but not by the mitogen-activated protein kinase kinase inhibitor, PD98059. Activation of Akt by carbachol was antagonized by atropine and inhibited by LY294002 and PP2. The Src-like tyrosine kinase inhibitor, PP2, also reduced carbachol stimulation of extracellular signal-regulated kinases 1/2 and cAMP-response element binding protein in a dose-dependent manner. Furthermore, carbachol increased tyrosine-phosphorylation of Fyn, a member of the Src-like tyrosine kinases. These results indicate that muscarinic acetylcholine receptors play an important role in oligodendrocyte progenitor survival through transduction pathways involving activation of Src-like tyrosine kinases and phosphatidylinositol 3-kinase/Akt.

Direct and Indirect Effects of Immune and Central Nervous System–Resident Cells on Human Oligodendrocyte Progenitor Cell Differentiation

In multiple sclerosis, successful remyelination within the injured CNS is largely dependent on the survival and differentiation of oligodendrocyte progenitor cells. During inflammatory injury, oligodendrocytes and oligodendrocyte progenitor cells within lesion sites are exposed to secreted products derived from both infiltrating immune cell subsets and CNS-resident cells. Such products may be considered either proinflammatory or anti-inflammatory and have the potential to contribute to both injury and repair processes. Within the CNS, astrocytes also contribute significantly to oligodendrocyte biology during development and following inflammatory injury. The overall objective of the current study was to determine how functionally distinct proinflammatory and anti-inflammatory human immune cell subsets, implicated in multiple sclerosis, can directly and/or indirectly (via astrocytes) impact human oligodendrocyte progenitor cell survival and differentiation. Proinflammatory T cell (Th1/Th17) and M1-polarized myeloid cell supernatants had a direct cytotoxic effect on human A2B5+ neural progenitors, resulting in decreased O4+ and GalC+ oligodendrocyte lineage cells. Astrocyte-conditioned media collected from astrocytes pre-exposed to the same proinflammatory supernatants also resulted in decreased oligodendrocyte progenitor cell differentiation without an apparent increase in cell death and was mediated through astrocyte-derived CXCL10, yet this decrease in differentiation was not observed in the more differentiated oligodendrocytes. Th2 and M2 macrophage or microglia supernatants had neither a direct nor an indirect impact on oligodendrocyte progenitor cell differentiation. We conclude that proinflammatory immune cell responses can directly and indirectly (through astrocytes) impact the fate of immature oligodendrocyte-lineage cells, with oligodendrocyte progenitor cells more vulnerable to injury compared with mature oligodendrocytes.

The PTEN inhibitor bisperoxovanadium enhances myelination by amplifying IGF-1 signaling in rat and human oligodendrocyte progenitors

Oligodendrocytes (OLGs) produce and maintain myelin in the central nervous system (CNS). In the demyelinating autoimmune disease multiple sclerosis, OLGs are damaged and those remaining fail to fully remyelinate CNS lesions. Therefore, current therapies directed to restrain the inflammation process with approaches that protect and reconstitute oligodendrocyte density would be essential to pave the way of myelin repair. A critical signal for oligodendrocytes is insulin‐like growth factor‐1 (IGF‐1), which promotes their development and ultimately myelin formation. PTEN inhibits the phosphoinositide 3‐kinase (PI3K)/Akt signaling, a convergence downstream pathway for growth factors such as IGF‐1. In this report, we temporarily inhibited PTEN activity by treating rat and human oligodendrocyte progenitors (OLPs) cultured alone or with dorsal root ganglion neurons (DRGNs) with bisperoxovanadium (phen). Our findings show that phen potentiates IGF‐1 actions by increasing proliferation of OLPs in a concentration‐dependent manner, and caused a sustained and time‐dependent activation of the main pathways: PI3K/Akt/mammalian target of rapamycin (mTOR) and MEK/ERK. At low concentrations, IGF‐1 and phen stimulated the differentiation of rat and human OLPs. Concordantly, the PTEN inhibitor together with IGF‐1 robustly augmented myelin basic protein accumulation in rat newborn and human fetal OLGs co‐cultured with DRGNs in a longer timeframe by promoting the elaboration of organized myelinated fibers as evidenced by confocal microscopy. Thus, our results suggest that a transient suppression of a potential barrier for myelination in combination with other therapeutic approaches including growth factors may be promising to improve the functional recovery of CNS injuries. GLIA 2013;62:64–77

Developmental regulation of α1A-adrenoceptor function in rat brain oligodendrocyte cultures

Abstract In this study, we examined the effect of norepinephrine (NE) on phosphatidylinositol-4,5-bisphosphate (PI) hydrolysis in progenitors and differentiated oligodendrocytes. NE caused a time- and concentration-dependent increase in total inositol phosphate (IPt) formation. The magnitude of this response increased as oligodendrocytes matured and was accompanied with an increase in α1-adrenoceptor (α1-AR) levels. To pharmacologically characterize the α1-AR subtype mediating PI hydrolysis in 12-day differentiated oligodendrocytes, various selective antagonists were used. Prazosin, the non-selective 1-AR antagonist, blocked NE-mediated IPt formation. Similarly, the α1A-AR selective competitive antagonists, 5-methyl urapidil (5-MU) and WB4104, were potent blockers of NE-mediated IPt formation. In contrast, the α1B- and α1D-AR antagonist, chloroethylclonidine and the α1D-AR antagonist, BMY 7378, had no effect. These results suggest that NE-induced PI hydrolysis in differentiated oligodendrocytes was mediated through the α1A-AR. Furthermore, this response was prevented by EGTA and CdCl2, suggesting a requirement for extracellular calcium. The presence of α1-AR subtypes in oligodendrocytes was confirmed by reverse transcriptase coupled polymerase chain reaction and by immunoprecipitation, with subtype specific antibodies. The results indicated that mRNA and protein for the α1A-, α1B- and α1D-AR subtypes were expressed. In conclusion, our findings show that oligodendrocytes express all three α1-AR subtypes but that only the α1A-AR was involved in NE-mediated IPt formation.

Regulation of miRNA 219 and miRNA Clusters 338 and 17-92 in Oligodendrocytes.

MicroRNAs (miRs) regulate diverse molecular and cellular processes including oligodendrocyte (OL) precursor cell (OPC) proliferation and differentiation in rodents. However, the role of miRs in human OPCs is poorly understood. To identify miRs that may regulate these processes in humans, we isolated OL lineage cells from human white matter and analyzed their miR profile. Using endpoint RT-PCR assays and quantitative real-time PCR, we demonstrate that miR-219, miR-338, and miR-17-92 are enriched in human white matter and expressed in acutely isolated human OLs. In addition, we report the expression of closely related miRs (miR-219-1-3p, miR-219-2-3p, miR-1250, miR-657, miR-3065-5p, miR-3065-3p) in both rodent and human OLs. Our findings demonstrate that miRs implicated in rodent OPC proliferation and differentiation are regulated in human OLs and may regulate myelination program in humans. Thus, these miRs should be recognized as potential therapeutic targets in demyelinating disorders.

Human Fetal Oligodendrocyte Progenitor Cells from Different Gestational Stages Exhibit Substantially Different Potential to Myelinate

To investigate age-related intrinsic regulation of the capacity of human fetal oligodendrocyte progenitor cells (OPCs) to myelinate, potential OPCs were selected from 15- to 23-gestational-week (gw) human fetal brain tissue based on the expression of gangliosides--recognized with the monoclonal antibody A2B5, which detects multipotent cells including OPCs--or platelet-derived growth factor receptor α (PDGFRα), an early marker of the oligodendroglial lineage. Cells were either cultured alone or cocultured with rat dorsal root ganglia neurons (DRGNs). When cultured alone, both the A2B5- and PDGFRα-selected cells exhibited age-dependent increases in early to mid-stage lineage markers, including sulfatides (O4 antibody) and the transcription factor Olig2, while the cell death rate correlated negatively with age. In coculture with neurons, cells also expressed the myelin components galactocerebroside (GC) and myelin basic protein (MBP), and ensheathed axons. In DRGN cocultures, A2B5+ cells derived from >19 gw produced more GC+/MBP+ cells compared with the 15-17-week cells. The number of GC+ cells making axonal contacts, and ensheathing axonal segments per cell increased proportionally to gestational age. This age-dependent difference in GC/MBP cell number and capacity to ensheath axons persisted when PDGFRα selection was used to enrich for the number of OPCs in cultures derived from younger ages. Addition of the growth factors brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) enhanced OPC differentiation under all conditions. These findings indicate that intrinsic regulatory mechanisms associated with the chronological age of the donor cells are key variables to assess when considering the myelination capacity of OPCs for cellular replacement therapy.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors

Significance Understanding of myelin diseases and development of new treatment options are at least partly hampered by the limited availability of human oligodendrocytes. Induced pluripotent stem cells (iPSC) may be an ideal tool to circumvent this problem; however, rapid and efficient protocols to generate oligodendrocytes from human iPSC are currently lacking. The induction of the transcription factors SOX10, OLIG2, and NKX6.2 in iPSC-derived neural progenitors accelerates oligodendroglial differentiation significantly resulting in up to 70% of O4+ oligodendrocytes within 28 d. These oligodendrocytes myelinate the CNS during development and after demyelination, and are suitable for pharmacological screens and disease modeling. The strategy presented herein will markedly facilitate the studying of human myelin diseases and the development of screening platforms for drug discovery. Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shi Rag−/− mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

Oligodendrogliopathy in Multiple Sclerosis: Low Glycolytic Metabolic Rate Promotes Oligodendrocyte Survival.

Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS. SIGNIFICANCE STATEMENT The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post‐mitotic neurons or differentiating neuronal precursors in vitro. In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non‐neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus‐enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN‐double positive population of cells might correspond to neuronal precursors. Although the NeuN‐positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo, but indicate that studies utilizing NeuN‐immunoreactivity as a definitive marker of post‐mitotic neurons in vitro should be interpreted with caution.