Tetsushi Kagawa

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

Stabilized β-Catenin Functions through TCF/LEF Proteins and the Notch/RBP-Jκ Complex To Promote Proliferation and Suppress Differentiation of Neural Precursor Cells

ABSTRACT The proliferation and differentiation of neural precursor cells are mutually exclusive during brain development. Despite its importance for precursor cell self renewal, the molecular linkage between these two events has remained unclear. Fibroblast growth factor 2 (FGF2) promotes neural precursor cell proliferation and concurrently inhibits their differentiation, suggesting a cross talk between proliferation and differentiation signaling pathways downstream of the FGF receptor. We demonstrate that FGF2 signaling through phosphatidylinositol 3 kinase activation inactivates glycogen synthase kinase 3β (GSK3β) and leads to the accumulation of β-catenin in a manner different from that in the Wnt canonical pathway. The nuclear accumulated β-catenin leads to cell proliferation by activating LEF/TCF transcription factors and concurrently inhibits neuronal differentiation by potentiating the Notch1-RBP-Jκ signaling pathway. β-Catenin and the Notch1 intracellular domain form a molecular complex with the promoter region of the antineurogenic hes1 gene, allowing its expression. This signaling interplay is especially essential for neural stem cell maintenance, since the misexpression of dominant-active GSK3β completely inhibits the self renewal of neurosphere-forming stem cells and prompts their neuronal differentiation. Thus, the GSK3β/β-catenin signaling axis regulated by FGF and Wnt signals plays a pivotal role in the maintenance of neural stem/precursor cells by linking the cell proliferation to the inhibition of differentiation.

Potentiation of Astrogliogenesis by STAT3-Mediated Activation of Bone Morphogenetic Protein-Smad Signaling in Neural Stem Cells

ABSTRACT Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.

Differentiation between dysmyelination and demyelination using magnetic resonance diffusional anisotropy

Using magnetic resonance (MR) diffusion-weighted method, we examined the optic and the trigeminal nerves of jimpy and twitcher mice, considered to be animal models of Pelizaeus-Merzbacher disease, hypomyelination disorder, and Krabbe disease, demyelination disorder, respectively. In jimpy mice, diffusional anisotropy of optic nerve did not show a significant difference compared to age-matched control mice, suggesting that diffusional anisotropy does exist in absence of multiple layers of myelin sheath. In twitcher mice, diffusional anisotropy was attenuated remarkably in the optic and trigeminal nerves. Loss of axonal straightness on longitudinal section confirmed by electron microscopy appeared to be the principal explanation for it. It is further suggested that this MR diffusion-weighted imaging method enables us to differentiate hypomyelination from demyelination in vivo.

Evidence for a second wave of oligodendrogenesis in the postnatal cerebral cortex of the mouse.

The existing view is that cortical oligodendrocytes (OLs) in rodents are born from the cortical subventricular zone (SVZ) after birth, but recent data suggest that many forebrain oligodendrocyte progenitor cells (OPCs) are specified much earlier (between E9.5 and E13.5 in the mouse) in the ventricular zone of the ventral forebrain under the control of sonic hedgehog (Shh) and migrate into the cortex afterward. We examined expression of specific early OL markers (PDGFRα, PLP/DM20, Olig2, and NG2) in the developing forebrain to clarify this issue. We propose that OPCs colonize the developing cortex in two temporally distinct waves. The gray matter is at least partially populated by a first wave of OPCs that arises in the medial ganglionic eminence and the entopeduncular area and spreads into the cortex via the developing cortical plate. The cerebral cortex benefits from the second wave of OPCs coming from residential SVZ. In the second wave, there might be two different types of precursor cells: PLP/DM20+ cells populating only inner layers and PDGFRα+ cells, which might eventually myelinate the outer regions as well.

Activation of Canonical Wnt Pathway Promotes Proliferation of Retinal Stem Cells Derived from Adult Mouse Ciliary Margin

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8‐week‐old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki‐67–positive proliferating cells in monolayer culture. The Wnt3a‐treated primary sphere cells were capable of self‐renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a‐treated cells showed nuclear accumulation of β‐catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki‐67–positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.

Dysregulation of axonal sodium channel isoforms after adult-onset chronic demyelination

Demyelination results in conduction block through changes in passive cable properties of an axon and in the expression and localization of axonal ion channels. We show here that adult‐onset chronic demyelination, such as occurs in demyelinating disorders and after nerve injury, alters the complement of axonal voltage‐dependent Na+ (Nav) channel isoforms and their localization. As a model, we used heterozygous transgenic mice with two extra copies of the proteolipid protein gene (Plp/−). Retinal ganglion cell axons in these mice myelinate normally, with young Plp/− and wild‐type mice expressing Nav1.2 at low levels, whereas Nav1.6 is clustered in high densities at nodes of Ranvier. At 7 months of age, however, Plp/− mice exhibit severe demyelination and oligodendrocyte cell death, leading to a profound reduction in Nav1.6 clusters, loss of the paranodal axoglial apparatus, and a marked increase in Nav1.2. We conclude that myelin is crucial not only for node of Ranvier formation, but also to actively maintain the proper localization and complement of distinct axonal Nav channel isoforms throughout life. The altered Nav channel isoform localization and complement induced by demyelination may contribute to the pathophysiology of demyelinating disorders and nerve injury.

In situ expression of PLP/DM-20, MBP, and CNP during embryonic and postnatal development of the jimpy mutant and of transgenic mice overexpressing PLP.

We analyzed by in situ hybridization the spatiotemporal expression of dm‐20, myelin basic protein (MBP) and 2′‐3′ cyclic nucleotide phosphodiesterase (CNP) during embryonic and postnatal development of the normal mouse and two plp/dm‐20 mutants: the jimpy mouse and a transgenic mouse overexpressing the plp gene. In the central nervous system (CNS) of the normal mouse, dm‐20 mRNA was detected at embryonic day (E)9.5 in the laterobasal plate of the diencephalon. The pattern of expression of CNP transcript was superimposable on that of dm‐20, but appeared slightly later, at E12.5. MBP mRNA was detected even later (E14.5), and, in addition, only in the caudal (rhombencephalon and spinal cord) territories of expression of dm‐20 and CNP. These observations support our previous proposals: (1) dm‐20‐expressing cells in the germinative neuroepithelium are precursors of oligodendrocytes, and (2) oligodendrocytes emerge from distinct pools of precursors along the neural tube (Timsit et al., 1995). In the jimpy mutant, despite the mutation in the plp gene, cells of the oligodendrocyte lineage developed normally. It is only at the time of myelin deposition that oligodendrocytes die. During embryonic development of the transgenic mutant overexpressing plp, there were no alterations in the spatiotemporal pattern or the level of expression of dm‐20 in the CNS, in contrast to the higher levels of dm‐20 observed in the peripheral nervous system (PNS). J. Neurosci. Res. 50:190–201, 1997.

Cell death of oligodendrocytes or demyelination induced by overexpression of proteolipid protein depending on expressed gene dosage

The transgenic mice, which were produced by introducing the wild type proteolipid protein (PLP) gene, revealed different neurological symptoms depending on expressed gene dosage. Homozygotes, which bore four more copies of the extra PLP gene, died before 4 weeks old with severe tremors and convulsions, while histologically hypomyelination and death of the oligodendrocytes were particularly noticeable. Heterozygotes, which bore two more copies and survived nearly 1 year, suffered from severe hindlimb palsy and bladder-rectal disturbances before death. These symptoms probably resulted from striking demyelination which occurred abruptly at a later stage of life. The heterozygous PLP-transgenic mice are thus available for a model of demyelination diseases.

Transgenic Systems in Studying Myelin Gene Expression

The myelin sheath is produced by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). It is necessary to introduce DNA into these cells to study gene regulation and the functions of myelin proteins. Since these cells are labile to commonly used techniques to introduce DNA, a transgenic system must be used for these studies. Myelin basic protein (MBP) and myelin proteolipid protein (PLP) are both highly abundant in CNS myelin, but are also produced in Schwann cells. Various lengths (6.5 kb to 256 bp) of the promoter region of the (classic) MBP gene directed oligodendrocyte-specific expression of the reporter gene in transgenic mice, but no expression was seen in Schwann cells. Promoter regions of the PLP gene (4.2-2.4 kb) seem to contain all the information required for correct spatiotemporal expression, but the level of expression was low. The first intron of the PLP gene is a candidate for the location of the enhancer-like element. Studies on MBP mutant mice carrying wild-type MBP gene or cDNA clearly demonstrated that one function of MBP is to make myelin lamellae compact by fusing the cytoplasmic surfaces of oligodendrocytes into the form of major dense lines. However, functional analysis of PLP gene products using a similar strategy produced confusing results. The wild-type PLP gene introduced into jimpy mutant mice (one of the PLP mutants) did not complement the mutant phenotype. Moreover, overexpression of the PLP gene itself (in wild-type background) was shown to produce a phenotype similar to that of the mutants, including arrest in oligodendrocyte maturation and hypomyelination. Thus, PLP gene products play a fundamental role in oligodendrocyte maturation as well as in the stabilization of myelin structure, and its expression must be tightly regulated.