Lynn D. Hudson

Alternative splicing accounts for the four forms of myelin basic protein

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

Identification of Dynamically Regulated microRNA and mRNA Networks in Developing Oligodendrocytes

MicroRNAs (miRNAs) play important roles in modulating gene expression at the posttranscriptional level. In postnatal oligodendrocyte lineage cells, the miRNA expression profile (“microRNAome”) contains 43 miRNAs whose expression dynamically changes during the transition from A2B5+ oligodendrocyte progenitor cells to premyelinating GalC+ cells. The combination of microRNAome profiling with analyses of the oligodendrocyte transcriptome reveals a target bias for a class of miRNAs which includes miR-9. We show that miR-9 is downregulated during oligodendrocyte differentiation. In addition, miR-9 expression level inversely correlates with the expression of its predicted targets, among which is the peripheral myelin protein PMP22. We found that PMP22 mRNA but not protein is detectable in oligodendrocytes, whereas Schwann cells producing PMP22 protein lack miR-9. We demonstrate that miR-9 interacts with the 3′ untranslated region of PMP22 and downregulates its expression. Our results support models in which miRNAs can act as guardians of the transcriptome.

Astrocytes Express Insulin-like Growth Factor-I (IGF-I) and Its Binding Protein, IGFBP-2, during Demyelination Induced by Experimental Autoimmune Encephalomyelitis

To assess the distribution of insulin-like growth-factor-related proteins during autoimmune CNS demyelination and remyelination, experimental autoimmune encephalomyelitis was produced by injecting Lewis rats with an emulsion containing guinea pig spinal cord and complete Freunds adjuvant. Tail weakness appeared at 10-12 days and was followed by hind and forelimb weakness. Paraplegia and incontinence were observed in some animals. From 8-40 days postinoculation (dpi), spinal cord sections were used to correlate lesion location and severity with mRNA distributions of insulin-like growth factor I (IGF-I), IGF-binding protein 2 (IGFBP-2), IGF-I-receptor (IGFR-I), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP). These were determined semiquantitatively by in situ hybridization. Fourteen dpi, there were inflammatory infiltrates and demyelination in both white matter (WM) and grey matter (GM). IGF-I and GFAP mRNAs were increased in these lesions and transcripts encoding myelin basic protein (MBP) were greatly reduced. Large lesions with extensive demyelination were evident in both WM and GM when mRNA levels of GFAP and IGF-I peaked 26 dpi. MBP mRNA levels began increasing 21 dpi and peaked 26 dpi, when a few thin regenerating myelin sheaths were found morphologically. Astrocytes, identified by their morphology and GFAP immunoreactivity, expressed very low levels of IGFBP-2 mRNA and peptide in normal controls; their levels were significantly higher 14 dpi, peaked 26 dpi, and then gradually decreased. Some neurons, as well as oligodendroglia in areas undergoing remyelination, expressed IGFR-I. Although levels of IGF-I, IGFBP-2, and GFAP mRNAs were highest in lesion areas, levels were also elevated around lesions and in some normal-appearing areas of WM and GM 14-40 dpi. The gene expression of both IGF-I and IGFBP-2 by hypertrophic GFAP-positive astrocytes was demonstrated 14-40 dpi by combined in situ hybridization and immunocytochemistry as well as by double immunostaining. Coexpression of IGF-I and IGFBP-2 in the same astrocyte was a frequent finding. Relative increases in both IGF-I, GFAP, IGFBP-2, IGFR-I, and MBP mRNAs peaked at about the same time. This suggests that during lesion progression and recovery, astrocytic expression of IGF-I-related peptides may reduce immune-mediated myelin injury. We also suggest that astrocytic IGFBP-2 in lesions may help target IGF-I to IGFR-I-expressing oligodendrocytes and promote remyelination of demyelinated axons.

Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system

The establishment and operation of the nervous system requires genetic regulation by a network of DNA‐binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys‐Cys‐His‐Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1‐like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303–321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system. J. Neurosci. Res. 50:272–290, 1997.

Myt1 family recruits histone deacetylase to regulate neural transcription.

The myelin transcription factor 1 (Myt1) gene family is comprised of three zinc finger genes [Myt1, Myt1L (Myt1‐Like) and NZF3] of the structurally unique CCHHC class that are expressed predominantly in the developing CNS. To understand the mechanism by which this family regulates neural differentiation, we searched for interaction partners. In both yeast and a mammalian two‐hybrid system, Myt1 and Myt1L interacted with Sin3B, a protein that mediates transcriptional repression by binding to histone deacetylases (HDACs). Myt1–Sin3B complexes were co‐immunoprecipitated from transfected mammalian cells and included HDAC1 and HDAC2. Myt1 and Myt1L could partner with all three Sin3B isoforms, the long form (Sin3BLF) that includes the HDAC‐binding domain, and the two short forms (Sin3BSF293 and Sin3BSF302) that lack this domain and may consequently antagonize Sin3BLF/HDAC‐mediated co‐repression. Myt1 or Myt1L interactions with the HDAC‐binding form of Sin3B conferred repression on a heterologous promoter. Oligodendrocytes were shown to express transcripts encoding each of the Sin3B isoforms. We present a model in which the Myt1 family of zinc finger proteins, when bound to a neural promoter, can recruit Sin3B. Depending on the relative availability of Sin3B isoforms, the Myt1 gene family may favor the silencing of genes during neural development.

Myelin transcription factor 1 (Myt1) modulates the proliferation and differentiation of oligodendrocyte lineage cells.

Myelin transcription factor 1 (Myt1) is a zinc finger DNA-binding protein that is expressed in neural progenitors and oligodendrocyte lineage cells. This study examines the role of Myt1 in oligodendrocyte lineage cells by overexpressing putative functional domains, a four-zinc finger DNA-binding region (4FMyt1) or a central protein-protein interaction domain (CDMyt1), without the predicted transcriptional activation domain. In the presence of mitogens, overexpression of 4FMyt1 inhibited proliferation of oligodendrocyte progenitors, but not cell types (astrocytes and NIH3T3 cells) lacking endogenous Myt1. Expression of 4FMyt1 inhibited the differentiation of oligodendrocyte progenitors into oligodendrocytes as assessed by morphology, immunostaining, and myelin gene expression. Progenitor differentiation was similarly inhibited by expression of CDMyt1 but only partially suppressed by overexpression of the intact Myt1. These data indicate that Myt1 may regulate a critical transition point in oligodendrocyte lineage development by modulating oligodendrocyte progenitor proliferation relative to terminal differentiation and up-regulation of myelin gene transcription.

Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation.

TPPP/p25, a recently identified tubulin polymerization‐promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. Here, we show that TPPP/p25 is strongly upregulated during the differentiation of primary oligodendrocyte cells as well as the CG‐4 cell line. The microRNA expression profile of CG‐4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR‐206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Overexpression of miR‐206 led to downregulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination.

A role for semaphorins and neuropilins in oligodendrocyte guidance

Oligodendrocytes develop in defined CNS regions as progenitor cells, which migrate to their final destinations, encountering soluble and membrane‐bound signals that influence their differentiation and potential to myelinate axonal projections. To identify the regulatory genes that may be involved in this process, microarray analysis of developing oligodendroglia was performed. Several neural guidance genes, including members of the neuropilin (NP) and semaphorin families were detected. These findings were verified and expanded upon using RT–PCR with RNA from fluorescent activated cell sorted A2B5+ oligodendrocyte progenitors and O4+ pro‐oligodendrocytes isolated from in vitro and in vivo sources. RT–PCR, western and immunocytochemical analyses revealed that oligodendrocytes expressed NP1, several alternatively spliced isoforms of NP2, and a broad spectrum of both soluble (Class 3), membrane‐spanning (Class 4–6), and membrane‐tethered (Class 7) semaphorin ligands. Class 3 semaphorins, in a modified stripe assay, caused the collapse of oligodendrocyte progenitor growth cones, redirection of processes, and altered progenitor migration. Our data support a role for neuropilins and semaphorins in orchestrating the migration patterns of developing oligodendrocytes in the CNS.

Nuclear organization in differentiating oligodendrocytes

Many studies have suggested that the 3D organization of chromatin and proteins within the nucleus contributes to the regulation of gene expression. We tested multiple aspects of this nuclear organization model within a primary cell culture system. Oligodendrocyte lineage cells were examined to facilitate analysis of nuclear organization relative to a highly expressed tissue-specific gene, proteolipid protein (PLP), which exhibits transcriptional upregulation during differentiation from the immature progenitor stage to the mature oligodendrocyte stage. Oligodendrocyte lineage cells were isolated from brains of neonatal male rodents, and differentiation from oligodendrocyte progenitors to mature oligodendrocytes was controlled with culture conditions. Genomic in situ hybridization was used to detect the single copy of the X-linked PLP gene within each interphase nucleus. The PLP gene was not randomly distributed within the nucleus, but was consistently associated with the nuclear periphery in both progenitors and differentiated oligodendrocytes. PLP and a second simultaneously upregulated gene, the myelin basic protein (MBP) gene, were spatially separated in both progenitors and differentiated oligodendrocytes. Increased transcriptional activity of the PLP gene in differentiated oligodendrocytes corresponded with local accumulation of SC35 splicing factors. Differentiation did not alter the frequency of association of the PLP gene with domains of myelin transcription factor 1 (Myt1), which binds the PLP promoter. In addition to our specific findings related to the PLP gene, these data obtained from primary oligodendrocyte lineage cells support a nuclear organization model in which (1) nuclear proteins and genes can exhibit specific patterns of distribution within nuclei, and (2) activation of tissue-specific genes is associated with changes in local protein distribution rather than spatial clustering of coordinately regulated genes. This nuclear organization may be critical for complex nucleic-acid—protein interactions controlling normal cell development, and may be an important factor in aberrant regulation of cell differentiation and gene expression in transformed cells.

Peripheral myelin protein 22 is regulated post-transcriptionally by miRNA-29a†

Peripheral myelin protein 22 (PMP22) is a dose‐sensitive, disease‐associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post‐transcriptionally regulated and the 3′untranslated region (3′UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post‐transcriptional level by targeting the 3′UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW‐body formation, the proposed cytoplasmic site for miRNA‐mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co‐regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22‐targeting miRNAs. Here we demonstrate that miR‐29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over‐expression of miR‐29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady‐state levels of PMP22. In contrast, inhibition of endogenous miR‐29a relieves the miRNA‐mediated repression of PMP22. Correlation analyses of miR‐29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post‐crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs.