miR-23 regulation of lamin B1 is crucial for ... · Disease Models & Mechanisms 179 Lamin B1 and miR-23in myelination RESEARCH ARTICLE uncover a novel function for a nuclear structural

RESEARCH ARTICLE

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INTRODUCTIONLamins are major components of the nuclear envelope and areessential for maintaining nuclear integrity, gene expression andmany other functions (Broers et al., 2006). Lamins can becategorized into two subfamilies (A and B types) that are encodedby different genes (LMNA and LMNB). LMNA mutations have beenlinked to a variety of diseases such as muscular dystrophy,cardiomyopathy, lipodystrophy and progeria (Capell and Collins,2006). To date, autosomal dominant leukodystrophy (ADLD) is theonly human disease that has been linked to an LMNB1 mutation(Padiath et al., 2006). In these patients, elevated levels of the laminB1 transcript and protein result from a duplication of the LMNB1gene. Post-mortem examination of the brains from ADLD patientsshowed severe myelin loss, although the axons in the white matterlesions were relatively spared from demyelination (Coffeen et al.,2000). In contrast to multiple sclerosis, oligodendrocytes arepreserved in ADLD and no signs of inflammatory infiltrate can bedetected. Nerve conduction velocity studies showed no evidenceof demyelinating features in the peripheral nervous systemsuggesting that LMNB1 duplications preferentially lead to myelinloss in the central nervous system (CNS).

Lamins provide anchorage sites for heterochromatin and thusepigenetically regulate transcription (Cohen et al., 2001). Specificmutations in LMNA that lead to familial partial lipodystrophy orthose that cause Hutchison-Gilford progeria syndrome causeaberrant localization of heterochromatin protein 1 in theperinuclear area, altered histone modification and mislocalizationof nuclear pore complexes (Scaffidi and Misteli, 2006; Shumakeret al., 2006). It is therefore suggested that LMNA mutations causethese disorders by altering the epigenetic regulation of genetranscription and perturbing trafficking across the nuclear

envelope. However, little is known about whether overexpressionof nuclear lamin can lead to alterations in the organization ofnuclear envelope components and of chromatin structure. Incontrast to manifestations of LMNA mutations in diverse tissues(Muchir and Worman, 2004), CNS demyelination is the onlyrecognized defect associated with the LMNB1 mutation (Padiathet al., 2006). Dominant-negative lamin B expression disruptsspindle assembly during mitosis (Tsai et al., 2006), suggesting thatlamin B functions actively in modulating mitotic organization.Homozygous truncated Lmnb1 in mice leads to defective lung andbone development and neonatal lethality, indicating theinvolvement of lamin B1 in development (Vergnes et al., 2004).Although a regulatory function of lamin B1 has been demonstratedin cell mitosis and embryonic organogenesis, little is known aboutits role in the developing CNS. The demyelination in ADLD isaccompanied by preservation of axons and oligodendrocytes andby decreased numbers of astrocytes with abnormal morphologysuggesting differential susceptibility of cell types to lamin B1overexpression. However, the connection between lamin B1 andglial development is unclear.

Recent progress in understanding small noncoding RNAs hasled to the identification of their regulatory roles in many biologicalfunctions (Kloosterman and Plasterk, 2006). MicroRNAs (miRNAs)have been implicated in normal physiological processes anddiseases (Stefani and Slack, 2008). In the developing CNS, manymiRNAs show a distinct expression pattern (Landgraf et al., 2007)supporting the idea that they might play important roles duringmammalian brain development, particularly in cell typedifferentiation in the CNS (Johnston and Hobert, 2003). Here, weinvestigated the mechanism by which elevated LMNB1 gene dosageleads to myelin loss in ADLD. We show that increased lamin B1expression results in disturbances in nuclear envelope organizationand nuclear pore transport. We demonstrate that the expressionlevel of lamin B1 is crucial in determining the progress ofoligodendrocyte maturation and myelin formation, and therefore

Disease Models & Mechanisms 2, 178-188 (2009) doi:10.1242/dmm.001065

miR-23 regulation of lamin B1 is crucial foroligodendrocyte development and myelinationShu-Ting Lin1 and Ying-Hui Fu1,*

SUMMARY

Duplication of the gene encoding lamin B1 (LMNB1) with increased mRNA and protein levels has been shown to cause severe myelin loss in thebrains of adult-onset autosomal dominant leukodystrophy patients. Similar to many neurodegenerative disorders, patients with adult-onset autosomaldominant leukodystrophy are phenotypically normal until adulthood and the defect is specific to the central nervous system despite the ubiquitousexpression pattern of lamin B1. We set out to dissect the molecular mechanisms underlying this demyelinating phenotype. Increased lamin B1expression results in disturbances of inner nuclear membrane proteins, chromatin organization and nuclear pore transport in vitro. It also leads topremature arrest of oligodendrocyte differentiation, which might be caused by reduced transcription of myelin genes and by mislocalization ofmyelin proteins. We identified the microRNA miR-23 as a negative regulator of lamin B1 that can ameliorate the consequences of excessive laminB1 at the cellular level. Our results indicate that regulation of lamin B1 is important for myelin maintenance and that miR-23 contributes to thisprocess, at least in part, by downregulating lamin B1, therefore establishing novel functions of lamin B1 and miR-23 in the regulation of oligodendrogliadevelopment and myelin formation in vitro.

1Department of Neurology, University of California San Francisco, San Francisco,CA 94158, USA*Author for correspondence (e-mail: [email protected])

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Disease Models & Mechanisms 179

Lamin B1 and miR-23 in myelination RESEARCH ARTICLE

uncover a novel function for a nuclear structural protein. We alsoidentified miR-23 as a negative regulator of lamin B1 that cancounteract the defects caused by increased lamin B1 dosage. Thisfinding allows us to place miR-23 and lamin B1 as new componentsin the regulatory networks of oligodendroglia biology and ofmyelin sheath formation and maintenance in vitro.

RESULTSLMNB1 overexpression affects localization of the nuclearmembrane protein LAP2 and chromatin organizationLamin B1 is ubiquitously expressed in various tissues (Broers etal., 1997), different parts of the brain (Padiath et al., 2006) and inboth neurons and glia (Fig. 1A). It interacts with nuclear andintegral membrane proteins including lamina-associatedpolypeptide (LAP2) and lamin B receptor (LBR) (Dreger et al.,2002). In order to examine the effects of LMNB1 overexpressionon the integrity of nuclear membrane proteins, ectopicoverexpression of LMNB1 was performed in neuronal (C17.2),astrocytic (SVG p12) and oligodendrocytic (N20.1) cell lines.Abnormal nuclear morphology such as extensive folding, blebbingand lobulation in the nuclear envelope was observed in all threecell types when LMNB1 was overexpressed. This abnormality didnot lead to significant changes in other nuclear membranecomponents in C17.2 and SVG cells (data not shown). Westernblot analysis revealed that LMNB1 (but not LMNA or LMNB2)overexpression leads to a reduction of LAP2 levels in N20.1 cells(Fig. 1B) and disrupts its co-localization with LMNB1 in thenuclear envelope (Fig. 1C). LBR co-localization and protein levelswere not affected by LMNB1 overexpression. In addition, noabnormality was observed for Emerin and MAN1 under theseconditions (data not shown). These results showed that LMNB1overexpression affects the subcellular localization and proteinlevels of LAP2 in oligodendrocytes.

Since abnormalities in nuclear morphology were induced byLMNB1 overexpression, we examined its effect on chromatinorganization. In fibroblasts overexpressing LMNB1,immunocytochemical analysis revealed that the localization ofheterochromatin protein 1 β (HP1β) and methylated histone 3(K3H9) were disrupted (Fig. 1D). The HP1β distribution in thenuclei of these cells was more distant from the nuclear envelopethan in control cells, indicating altered chromatin organization.

LMNB1 overexpression suppresses oligodendrocyte-specific genesNuclear lamins interact directly with heterochromatin and regulateDNA synthesis and transcription. In addition, LAP2 is involved inregulating gene expression (Nili et al., 2001). We therefore soughtto determine whether LMNB1 overexpression affects transcriptionin a cell-type-specific manner in the CNS. Luciferase reporteranalysis revealed that LMNB1 overexpression did not change thetranscription from the neurogenic differentiation 1 (NeuroD)(neuron specific) promoter (Fig. 2A) but increased transcriptionfrom the GFAP (astrocyte specific) promoter (Fig. 2B). In addition,LMNB1 overexpression significantly repressed transcription fromthe oligodendrocyte-specific promoters of myelin basic protein(MBP), proteolipid protein (PLP) and myelin oligodendrocyteglycoprotein (MOG) (Fig. 2C-E). Therefore, LMNB1 overexpressionexhibits direct effects on transcriptional regulation in anoligodendrocyte- and astrocyte-specific manner.

Fig. 1. LMNB1 overexpression affects localization of the nuclearmembrane protein LAP2 and chromatin organization. (A) Dual-immunostaining of Lmnb1 (LB1) (green) and NeuN (neuronal nuclei, a neuronmarker), GFAP (glial fibrillary acidic protein, an astrocyte marker) or CNP (cyclicnucleotide 3‘ phosphodiesterase, an oligodendrocyte marker) (all red) in brainsections of C57BL/6J mice at postnatal day 16. (B) Western blots (left panel) ofoligodendrocyte cells (N20.1) overexpressing a green fluorescent protein (GFP)vector, a GFP-LMNA fusion protein (LA), a GFP-LMNB1 fusion protein (LB1) or aGFP-LMNB2 fusion protein (LB2). Antibodies against LB1, LAP2, LBR andGAPDH were used. Quantifications of the LAP2 bands from the western blotare shown in the panel on the right. (C) Immunostaining of LAP2 (red, upperpanel) or LBR (red, lower panel) in N20.1 cells overexpressing GFP vector orGFP fusion constructs (LA, LB1 or LB2). (D) Three examples of immunostainingwith methylated histone 3 (K3H9) (green) and HP1β (red) in culturedfibroblasts. The white arrowhead points to blebbing in the nuclear envelope.LB1=LMNB1 overexpression. Bars, 20 μm (A); 10 μm (C,D).

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Lamin B1 and miR-23 in myelinationRESEARCH ARTICLE

LMNB1 overexpression affects nuclear exportLamins have been reported to associate with the nuclear poreproteins and affect their recruitment and maintenance in thenuclear pore complex (Smythe et al., 2000). To assess nucleartransport following LMNB1 overexpression, we compared relativeamounts of glutathione S-transferase (GST)-GFP proteins thatwere fused to either a nuclear localization sequence (NLS) derivedfrom the simian virus 40 (SV40) T antigen or a nuclear exportsequence (NES) derived from HIV Rev. The GST-GFP fusionproteins were large enough in size that they could not freely diffusethrough the nuclear pore. GST-GFP fusion proteins containing theNLS (NLS-GFP) or the NLS plus NES (NLS-GFP-NES) wereintroduced into HEK293 cells with or without a cDNA constructexpressing LMNB1 or LMNB2. The cytoplasmic and nuclear levelsof GST-GFP were then assessed by western blot analysis. NLS-GFP accumulated equally in the cytoplasm and nuclei of cells co-transfected with either a vector or the cDNA construct expressingLMNB1 or LMNB2 (Fig. 3A,B). In contrast, NLS-GFP-NES proteinaccumulated in higher levels in the nuclei of cells co-transfectedwith the cDNA construct expressing LMNB1 compared with inthe nuclei of cells transfected with either the vector or the cDNAconstruct expressing LMNB2 (Fig. 3B). NLS-GFP-NES proteinlevels in the cytoplasmic fractions were similar for all threeconstructs (Fig. 3A). To further examine whether the increasedaccumulation of NLS-GFP-NES protein in nuclei was the resultof enhanced nuclear import and/or impaired nuclear export

following LMNB1 overexpression, the cells were treated withleptomycin B (LMB, an inhibitor of nuclear export in HIV Rev).In the presence of LMB, nuclear NLS-GFP-NES protein levels weresimilar in cells co-transfected with the vector control or with thecDNA construct expressing LMNB1 or LMNB2, suggesting thatLMNB1 overexpression impairs nuclear export. Furthermore,western blot analysis of whole cell lysates showed that LMNB1overexpression also resulted in reduction of Nup153 (Fig. 3C), anucleoporin that interacts with lamin B (Smythe et al., 2000). Takentogether, these results demonstrate that LMNB1 overexpressionimpairs nuclear-cytoplasmic shuttling and leads to reducedNup153 levels.

Lamin B1 is developmentally regulatedAlthough lamin B1 is widely expressed in various parts of the brain(Allen Brain Atlas, www.brain-map.org), its temporal expressionpattern has not been studied. In addition, ADLD is an adult-onsetdisease; patients have no symptoms until the forth or fifth decadesof life. We set out to examine the temporal expression profile oflamin B1 during mouse brain development using quantitative RT-PCR and western blotting. mRNA and protein levels of lamin B1peak at birth or postnatal day 1, followed by a gradual decreasefrom postnatal day 1 to 10 months of age (Fig. 4A,B). Therefore,lamin B1 is developmentally regulated. This is consistent with theprevious finding that Lmnb1 mRNA levels are graduallydownregulated during oligodendrocyte maturation in vitro (Dugas

Fig. 2. LMNB1 overexpression suppressesoligodendrocyte-specific genes. Luciferasereporter assays for the (A) NeuroD promoter inneuronal (C17.2) cells, (B) GFAP promoter in glial(N20.1) cells, and the (C) MBP, (D) PLP and (E)MOG promoters in glial (N20.1) cells. Relativepromoter activities for cells co-transfected witheither a vector or a construct containing cDNAencoding LMNB1 (LB1) are shown. Cells weregrown at 34°C for (B) or 39°C for (C-E). *P<0.05,**P<0.01.

Fig. 3. LMNB1 overexpression affects nuclear export. (A) Western blots of cytoplasm-enriched fractions from HEK293 cells overexpressing NLS-GFP (left panel)or NLS-GFP-NES (right panel). These cells were co-transfected with vector or with a flag-tagged cDNA construct encoding either LMNB1 (LB1) or LMNB2 (LB2).GAPDH was used as a loading control and HDAC1 was used as a fraction control. (B) Western blots of nuclear-enriched fractions from cells overexpressing NLS-GFP (left panel) or NLS-GFP-NES (middle and right panels). Cells were treated with leptomycin B (LMB) (20 ng/ml) for 2 hours before protein extraction and thenuclear-enriched fractions were used for western blot analysis (right panel). HDAC1 was used as a loading control and GAPDH was used as a fraction control.(C) Western blot with antibody against Nup153 for whole cell extracts from N20.1 cells that were transfected with vector alone or with a cDNA constructencoding LMNB1.

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et al., 2006). Interestingly, the expression pattern of Lmnb1 showsa reverse correlation with other myelin-specific proteins [CNP, MBPand MAG (myelin-associated glycoprotein)] (Fig. 4B), suggestinga possible role for Lmnb1 in the regulation of oligodendrocytedevelopment.

Lamin B1 is regulated by miR-23miRNAs play essential roles in the development of variousorganisms from nematodes to mammals. To test whether miRNAsplay a role in the developmental regulation of lamin B1, we soughtto identify miRNAs that target mouse Lmnb1 mRNA. The 3�untranslated region (UTR) of Lmnb1 mRNA was scanned forpotential miRNA binding sites. Five sites were chosen because oftheir conservation in human, chimpanzee, mouse, rat, dog andchicken genomes (supplementary material Fig. S1A). The Lmnb13�UTR was fused to the luciferase reporter in an expressionplasmid before co-transfection into HEK293 cells with each ofthese miRNAs to examine their repressive effect. Transfection ofmiR-23a and miR-23b resulted in significantly reduced luciferaseactivities (Fig. 5A). Western blot was used to reveal thatendogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells. No change in LMNB1 mRNA levelswas noted (data not shown). In addition, knocking downendogenous miR-23 with synthetic antisense oligonucleotidesresulted in increased LMNB1 levels (Fig. 5C). Furthercharacterization of the Lmnb1 3�UTR revealed that there are three

separate miR-23 binding sites within this region (seesupplementary material Fig. S1 and Table S1). The codingsequences of the GFP-Lmnb1 fusion protein were then fused toeither a wild-type Lmnb1 3�UTR or a Lmnb1 3�UTR with mutatedmiR-23 sites to examine the accessibility of miR-23 to the 3�UTRof Lmnb1 mRNA. Introduction of these GFP-Lmnb1 plasmids intoHEK293 cells led to nuclear envelope localization (data notshown). When the wild-type 3�UTR construct was transfected,addition of miR-23a or miR-23b into the system resulted in asignificant reduction in the GFP-Lmnb1 levels (Fig. 5D). The GFP-Lmnb1 level was not affected in cells transfected with the mutated3�UTR construct, indicating that the miR-23-binding sites inLmnb1 3�UTR are authentic. We next examined the temporalexpression pattern of miR-23 in the mouse brain. Northernblotting revealed that miR-23a and miR-23b are similarly expressedin the brain and that their levels gradually increase with age in apattern that is complimentary to that of Lmnb1 (Fig. 5E,F). Takentogether, these results indicate a role for miR-23 in downregulatingLmnb1 expression, especially in adulthood.

Fig. 4. Lamin B1 is regulated developmentally. (A) qPCR of Lmnb1 mRNAfrom C57BL/6J mouse brain tissue at the indicated ages. Means from a singlerepresentative experiment measured in triplicate are shown. Twoindependent experiments were performed. (B) Western blot analysis of totalprotein lysates from C57BL/6J mouse brain at the indicated ages.Immunoblots were performed using antibodies against Lmnb1 (LB1), NeuN,GFAP, CNP, MBP and MAG. GAPDH was used as a loading control.

Fig. 5. Lamin B1 is regulated by miR-23. (A) Luciferase activity of the reportergene fused with the Lmnb1 3�UTR in the presence of the indicated miRNA. Thedata represent the mean value from three independent experiments ± s.e.m.(B) Western blot analysis of endogenous LMNB1 levels in lysates from HEK293cells transfected with constructs expressing the indicated miRNAs. (C) Westernblot analysis of LMNB1 from HEK293 cells transfected with the indicated anti-sense oligonucleotides. (D) Western blot analysis of Lmnb1 from HEK293 cellsco-expressing both miR-23 and the GFP-Lmnb1 coding sequence fused toeither a wild-type or mutant Lmnb1 3�UTR. All three miR-23 binding sites werealtered in the mutant 3�UTR. An antibody against GFP was used to reveal thelevel of GFP-Lmnb1. (E) Northern blot analysis of RNA isolated from C57BL/6Jmouse brain at the indicated ages. The probes used were miR-23a, miR-23band U6 small nuclear (sn)RNA. (F) Levels of the miR-23a transcript and theLmnb1 protein in the C57BL/6J mouse brain at the indicated ages.

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The effects of lamin B1 and miR-23 on oligodendrogenesisOverexpression of LMNB1 or miR-23 in glial cultures wasperformed to test the roles of lamin B1 and miR-23 in glialdevelopment. To achieve miR-23 or LMNB1 overexpression, weused a lentiviral vector that permits efficient expression ofexogenous miR-23 or LMNB1 in mouse primary glial cultures.Overexpression of miR-23 led to significantly increased numbersof cells with positive immunoreactivity to CNP (Fig. 6A,B) and MBP(data not shown). This increase was accompanied by increasedlevels of CNP (Fig. 6C) and MBP (data not shown) suggesting thatmiR-23 causes enhanced oligodendrogenesis. In contrast, LMNB1overexpression caused a reduction in the numbers of CNP- or MBP-positive cells and decreased CNP and MBP protein levels, implying

a repressive effect of LMNB1 on oligodendrogenesis. Neither miR-23 nor LMNB1 overexpression exerted significant effects on GFAPexpression in mixed glial cultures. These results indicate thatoligodendrocyte development is specifically suppressed by laminB1 and enhanced by miR-23. miR-23 and LMNB1 were alsoindividually expressed in cultured astrocytes or oligodendrocytesthat had been purified from mouse brains. Consistent with previousresults, miR-23 enhanced, and LMNB1 repressed, oligodendrocytedifferentiation in oligodendrocyte-enriched cultures. Neither miR-23 nor LMNB1 overexpression caused any alteration in GFAPimmunoreactivity in astrocytic glial cultures (data not shown).

Regulation of oligodendrocyte development is a complexbiological process that involves intricate transcriptional regulationand many signaling pathways among neurons, astrocytes andoligodendrocytes. Astrocytes are very abnormal in postmortemADLD brains – one possibility is that oligodendrocytedifferentiation could be regulated by factors secreted by astrocytes.To address this possibility, conditioned media from astrocytesoverexpressing miR-23 or LMNB1 was used to culture mixed glialcells. This media did not affect oligodendrocyte differentiation inprimary cultures (as assessed by immunocytochemistry andWestern blot) (supplementary material Fig. S2), indicating that theobserved differential effect of miR-23 and LMNB1 on culturedoligodendrocytes was not mediated by soluble factors secreted fromastrocytes but by the direct effects of miR-23 and LMNB1. Indeed,immunocytochemical analysis in purified oligodendrocyteprogenitor cells (OPC) recapitulates our previous findings in mixedglial cultures (Fig. 6D). We concluded that miR-23 acts as a positiveregulator of oligodendrocyte differentiation whereas LMNB1 is asuppressor of oligodendrocyte maturation, raising the possibilitythat the finely regulated expression of LMNB1 might be importantin the control of oligodendrocyte development and myelinformation in vivo.

Lamin B1 overexpression leads to altered MBP and PLP subcellularlocalizationThe process of OPC differentiation into myelinatingoligodendrocytes occurs in distinct temporal stages. To gain moreinsight into whether the lamin B1-mediated inhibitory effect onoligodendrogenesis is specific to certain developmental periods,immunocytochemical experiments were carried out with stage-specific markers. These antigenic characterizations were conductedin OPC cultures with proliferation (+PDGF) or differentiation (+T3)medium. LMNB1 overexpression did not alter the pattern of NG2(neural-glial antigen 2) staining but did cause decreases both inthe number of branched processes visualized by galactocerebroside(GalC) and in CNP immunoreactivity (Fig. 7A). In control cells,the presence of MBP and PLP was distributed in the cell bodiesand distal cellular processes, whereas in LMNB1-overexpressingcells the majority of MBP was found in the cell nuclei and PLP waslocalized to perinuclear regions and proximal portions of processes.The repressive effect of LMNB1 overexpression was also reflectedin the reduced levels of CNP and the myelin proteins MBP, PLPand MAG (Fig. 7B). MBP immunoreactivity was used to assessprocess branching using Sholl analysis, a quantitative method forradial distribution of dendritic morphology and complexity (Fig.7C). There was a dramatic decrease in the number of intersectionsof processes in oligodendrocytes overexpressing LMNB1 compared

Fig. 6. The effects of lamin B1 and miR-23 on oligodendrogenesis.(A) Immunostaining of CNP (red, upper panel) or GFAP (red, lower panel) in 14-day-old in vitro mixed glia cultures infected with vector, miR-23 or the cDNAconstruct encoding LMNB1. Infected cells are indicated in green by GFP.(B) Average fractions of CNP-positive and GFAP-positive cells. The totalnumbers of cells were determined by DAPI staining. Data are presented asratio ± s.e.m. from three independent experiments. *P<0.05, †P<0.5, ††P<0.01.The P values for CNP and GFAP are 0.0045 and 0.1477, respectively. (C) Westernblot analysis of CNP, GFAP and GAPDH levels in protein lysates from mixed glialcultures overexpressing miR-23 or the cDNA construct encoding LMNB1.(D) Immunostaining of MBP (red) in purified OPCs infected with vector, miR-23or the cDNA construct encoding LMNB1. Similar experiments with the cDNAconstruct encoding mouse Lmnb1 gave the same results (data not shown).Cells were cultured in differentiation medium for 5 days. Infected cells areindicated in green by GFP. Bars, 20 μm (A); 10 μm (D).

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with in control cells. To determine whether the aberrant localizationof MBP and PLP was the result of a defect in process formation,immunocytochemistry was conducted using an antibody againstMAG, which correlates with late stage myelinatingoligodendrocytes. MAG was present in the distal branchingprocesses of oligodendrocytes that overexpressed LMNB1 despitetheir reduced branching morphology versus control cells (Fig. 7A).These experiments were also carried out by overexpressing mouseLmnb1 and similar results were obtained (data not shown). Thesedata suggest that lamin B1 overexpression can alter the subcellularlocalization of MBP/PLP and reduce the branching of maturingoligodendrocytes.

Lamin B1 overexpression leads to reduced oligodendrocyte- andmyelin-specific proteins in vivoTo further validate our finding that lamin B1 can inhibitoligodendrocyte maturation in vivo, we obtained brain tissues fromtwo ADLD patients. Protein extracts from these brain tissues wereused in western blotting and the levels of oligodendrocyte- (CNP)and myelin- (MBP, PLP, MAG) specific proteins were determinedwith specific antibodies. In agreement with our in vitro finding,the levels of these proteins were all reduced in ADLD brain tissueswhen compared with control brain tissue (Fig. 7D). In addition, thelevels of reduction were greater in tissues with higher lamin B1overexpression (Fig. 7E), supporting a dosage effect on theoligodendrocyte/myelin inhibition by lamin B1.

miR-23 can rescue defects caused by lamin B1 overexpression inoligodendrocytesSince miR-23 suppresses lamin B1 and exhibits positiveregulation of oligodendrocyte maturation, we investigatedwhether miR-23 could rescue the oligodendrocyte maturationdefects caused by lamin B1 overexpression. First, we set out todetermine the dosage effect of miR-23 on Lmnb1 expression bytransfecting HEK293 cells with various ratios of miR-23 and theconstruct expressing GFP-tagged Lmnb1. Western blot analysisrevealed a dose-dependent repressive effect of miR-23 on Lmnb1expression (Fig. 8A). Notably, at a dose ratio of 50:1, miR-23reduced both the exogenous Lmnb1 (GFP-LB1) and endogenousLMNB1 (LB1) proteins. We then monitored the extent ofoligodendrocyte differentiation under different dosecombinations using anti-MBP immunocytochemistry. Noalteration in MBP nuclear localization was observed when co-expressing miR-23 with the construct expressing GFP-Lmnb1 ata ratio of 1:1 compared with co-transfecting a vector controlinstead of miR-23 (Fig. 8B). At an expression ratio of 10:1, cellularprocesses were clearly visible but reduced in number. At a higherexpression ratio of 50:1, a substantial (although not complete)rescue of MBP distribution in oligodendrocytes was found.Increasing amounts of miR-23 enhanced this rescue indicatingthat this is a dosage-dependent biological effect. In contrast,when constructs carrying Lmnb1 with mutated miR-23 bindingsites were used, no rescue was found even at a dose ratio of 50:1

Fig. 7. Lamin B1 alters MBP and PLPdistribution patterns. (A) Mouse OPCswere infected with a GFP vector (control)or the construct expressing GFP-LMNB1(LB1) and were then cultured inproliferation (+PDGF) or differentiationmedium (+T3) for 5 days.Immunocytochemistry ofoligodendrocytes was performed withantibodies against NG2, GalC, CNP, MBP,PLP and MAG (red). (B) Western blotanalysis of CNP, MBP, PLP, MAG andGAPDH expression in protein lysates fromthe OPC cultures described in (A).(C) Sholl analysis of oligodendrocytesoverexpressing the GFP vector (whitebars) or the GFP-LMNB1 construct (blackbars). Shell radius indicates the distancesfrom the cell body. Error bars represents.e.m. (n=7 for vector control, n=8 forLB1). (D) Western blot analysis of LMNB1(LB1), CNP, MBP, PLP, MAG and GAPDHexpression in protein lysates from thebrain tissues from two ADLD brains (Pt1and Pt2) and one normal control brain.(E) Protein quantities were plotted inrelation to the quantities of LMNB1 in (D).

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(Fig. 8C,D). In agreement with our previous observation, co-expression of miR-23 and the GFP-Lmnb1 construct carryingeither the wild-type or the mutant Lmnb1 3�UTR did not alterthe MAG immunoreactivity in distal oligodendrocyte processes.Together, these findings suggest that increased expression oflamin B1 causes premature arrest of oligodendrocyte maturationand that miR-23 can enhance oligodendrocyte development bysuppressing lamin B1.

DISCUSSIONLamin B1 is one of the structural proteins in the nuclear envelope.Low-level overexpression of structural proteins is usually tolerableand does not cause significant pathological consequences (Abe andOshima, 1990). However, high levels of overexpression can resultin detrimental cellular effects such as overloaded protein traffickingor degradation systems (Garbern, 2007). Increased gene dosageaccompanied by excessive gene products has been reported inassociation with many neurodegenerative diseases (Singleton et al.,2003; Rovelet-Lecrux et al., 2006). Excessive LMNB1 productionowing to LMNB1 duplication is associated with the CNSleukodystrophy phenotype in ADLD patients, demonstrating thatmyelin genesis and/or maintenance is sensitive to lamin B1 levels(Padiath et al., 2006). In this study, we showed that LMNB1overexpression caused abnormal nuclear envelope morphology andaltered the expression and distribution of LAP2. Overexpressionof B-type lamins has been shown to promote nuclear membranegrowth and intranuclear membrane formation in amphibianoocytes and epithelia and in mammalian kidney cells in a CaaX-motif-dependent manner (Prufert et al., 2004; Ralle et al., 2004).In these systems, expression of different lamins at moderate levelsinduced nuclear envelope growth whereas expression at higher

levels led to the formation of intranuclear membranes. Examinationby electron microscopy showed that these intranuclear membraneswere not continuous with the inner nuclear membrane and weredevoid of pore complexes. In agreement with these findings, ourectopic overexpression of LMNB1 in established neuronal and glialcells increased the surface area of the nuclear membrane and thenumber of intranuclear aggregates. One possibility is that LMNB1overexpression, through formation of these intranuclearmembranes, leads to the altered subcellular localization of LAP2and perturbed nuclear transport. It is noteworthy that in theprimary culture system, lentivirus induces moderate overexpressionof LMNB1 in oligodendrocytes leading to differentiation defectsand drastic effects on myelin protein expression accompanied byminimal nuclear envelope distortion and growth when comparedwith established culture systems.

Loss of function owing to truncated Lmnb1 leads to postnatallethality with defective lung and bone development in mice(Vergnes et al., 2004), however it is not known whether thistruncation is associated with any neurological deficits in the brain.Organization of the nuclear genome in relation to the nuclearlamina plays an important role in the regulation of gene expression(Taddei, 2007). Fibroblasts cultured from Lmnb1 knockout miceexhibit drastic changes in the transcription level of 498 genes(Malhas et al., 2007). Moreover, Drosophila lamin B has been foundto interact directly with about 500 genes that are clustered in thegenome and are developmentally expressed (Pickersgill et al.,2006). The alteration in chromatin organization that we observedhere could also be attributed to the aberrant intranuclear membraneformation caused by LMNB1 overexpression (Prufert et al., 2004;Ralle et al., 2004). These changes might then lead to alterations intranscriptional regulation and DNA replication (Somech et al.,

Fig. 8. miR-23 can rescue defects caused by laminB1 overexpression in oligodendrocytes. (A) LaminB1 levels in HEK293 cells transfected with differentratios of miR-23 and the construct expressing GFP-Lmnb1. LB1 indicates the endogenous LMNB1 andGFP-LB1 represents the exogenous GFP-Lmnb1.(B) Immunocytochemistry of GFP (green) and MBP(red) in mouse oligodendrocytes co-transfectedwith miR-23 and the construct expressing GFP-Lmnb1 at the indicated ratios. Cells were cultured indifferentiation medium for 4 days. (C) Lmnb1 levelsin HEK293 cells co-transfected with miR23 and theGFP-Lmnb1 construct containing either the wild-type (WT) or the mutated (Mut) Lmnb1 3�UTR at theindicated ratios. (D) Immunocytochemistry of MBPor MAG in mouse oligodendrocytes co-transfectedwith miR-23 and the GFP-Lmnb1 constructcontaining either the wild-type or the mutantLmnb1 3�UTR at a ratio of 50:1. Cells were cultured indifferentiation medium for 4 days. Bars, 10 μm.

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2007). Cells with different specifications have distinct distributionsignatures of chromosome territories and pericentromericheterochromatin (Mayer et al., 2005). Distinct gene expressionpatterns have been demonstrated in neurons, astrocytes andoligodendrocytes despite their identical genomic structure (Cahoyet al., 2008). Indeed, the specific effects of LMNB1 overexpressionon gene transcription were confirmed by the repression of myelin-specific genes and the activation of GFAP transcription. Theseresults suggest that overexpression of LMNB1 could disturb theunique expression patterns in individual CNS cell types and thatphenotypes would only appear in certain cell types that arevulnerable to the radical transcriptional changes.

Despite the ubiquitous spatial expression in neuronal and glialcell types, the temporal expression of lamin B1 is regulated suchthat the pattern is complementary to that of the majoroligodendrocyte and myelin proteins (including CNP, MBP andMAG). Lamin B1 overexpression exerts inhibitory effect on thegenesis of oligodendrocytes. In contrast, ectopic expression ofmiR-23, a negative regulator of lamin B1, can enhanceoligodendrogenesis. These results established miR-23 and laminB1 as putative regulators in oligodendroglia development andmyelin sheath formation. To date, accumulating reports provideevidence that miRNAs function in the regulation of manyphysiological processes such as neuronal development, cell-fatespecification, differentiation and synaptogenesis (Schratt et al.,2006; Visvanathan et al., 2007). In the presence of excess miR-23,a greater proportion of cells express mature oligodendrocytemarkers and increased levels of mature myelin proteins togetherwith having a multipolar morphological appearance, indicating thatmiR-23 can enhance oligodendrogenesis. In contrast, excessivelamin B1 leads to lower numbers of cells expressing mature markerswith reduced levels of mature myelin proteins, suggesting defectivedifferentiation of oligodendrocytes. These in vitro findings will needto be validated in an in vivo system. One piece of evidencesupporting the in vivo relevance is that we observed reduced levelsof oligodendrocyte- and myelin-specific proteins in ADLD braintissues. It is possible that miR-23 can also enhance oligodendrocytedevelopment through other lamin B1-independent pathways. Inthis case, excessive lamin B1 production in the cells might sequestermiR-23 during maturation, thereby further adding to thedeteriorating myelin loss that results from lamin B1 overexpressionin the CNS. Since many transcription factors and environmentalcues are involved in the complex control mechanisms ofoligodendrogenesis and myelin sheath formation, identification ofpossible, additional downstream targets of miR-23 inoligodendrogenesis pathways might provide new insight into themechanisms of oligodendrocyte development, myelin formationand maintenance.

In oligodendrocytes, increased LMNB1 gene dosage causedpremature arrest of differentiation with the principal phenotypebeing a lack of MBP and PLP at the cell surface. The absence ofMBP and PLP induced by lamin B1 overexpression is specific sinceMAG, a myelin protein sharing the same biosynthetic pathway asPLP, was present on the cell surface of oligodendrocytes. Thedecreased appearance of MBP and PLP at branching processescould be caused by lower protein levels owing to suppressedtranscription. It is also possible that defective nuclear exportcontributes to this phenomenon. MBP is severely affected by lamin

B1 overexpression and the lack of functional MBP results in a lackof myelin in mouse brain tissue. Re-introducing MBP into thismutant mouse improved the degree of myelin assembly and majordense line formation (Popko et al., 1987; Readhead et al., 1987).Because MBP can be transported between the cytoplasm and thenucleus (Pedraza et al., 1997), and because of its distribution in theoligodendrocyte nuclei, soma and myelin sheathes, it might havea regulatory role in gene transcription and oligodendrocyteprocessing during myelination (Verity and Campagnoni, 1988). Theregulatory role of MBP is supported by an increased level of MBPin the nuclei and cytoplasm of oligodendrocytes undergoingmyelinogenesis or myelin maintenance; in addition, plasmalemmalMBP occurs in quiescent oligodendrocytes (Hardy et al., 1996). Toa lesser extent, lamin B1 overexpression also disrupted theappearance of PLP in distal processes of oligodendrocytes. PLPfunctions in maintaining the adhesion and stabilization of theextracellular surfaces of the myelin sheath (Klugmann et al., 1997).Interestingly, PLP is overexpressed in classical Pelizaeus-Merzbacher disease (PMD) and, instead of reaching the cell surface,mutated PLP is aberrantly retained in the endoplasmic reticulum,Golgi apparatus or nuclear envelope in X-linked spastic paraplegia(Koizume et al., 2006). These mutations induce in vivohypomyelination, arrest oligodendrocyte maturation and lead todeath of oligodendrocytes (Kagawa et al., 1994; Readhead et al.,1994). Despite the similarity in both the aberrant subcellularlocalization of PLP and the developmental defects inoligodendroglia, lamin B1 overexpression does not result in thedemise of oligodendrocytes, suggesting that the myelin defectsinduced by lamin B1 overexpression are fundamentally distinctfrom those caused by mutations in the myelin genes. Theappearance of normal numbers of NG2 cells, which aremorphologically normal, suggests that lamin B1 overexpressiondoes not affect cell specification and patterning during the earlydevelopment of oligodendrocytes. Our results indicate that thedevelopmental defects induced by lamin B1 overexpression occurmainly during terminal differentiation. It is possible that a reductionin MBP and PLP assembled into the myelin membrane can makethe sheath less compact and more unstable, therefore leading toaccelerated myelin breakdown which is reflected by the observationof demyelination, without death of the oligodendrocytes, in thebrains of ADLD patients.

We showed that the defects induced by lamin B1 overexpressioncan be reversed by miR-23 in a dosage-dependent mannerdemonstrating that lamin B1 has a threshold effect onoligodendrocyte development and myelin production. It is notsurprising that a threshold effect can apply to normal myelinproteins since dysmyelination and demyelination occur withincreased or decreased dosage of myelin proteins such as PLP andPMP22 (Lupski et al., 1991; Inoue et al., 1996). Unexpectedly, theubiquitously expressed nuclear lamina protein, lamin B1, potentlyinfluences the amount and quality of myelin formation in the CNS.miR-23 is important for the process of transitioning ofoligodendrocyte progenitors into mature oligodendrocytes, whereit acts, at least in part, by antagonizing lamin B1 levels. Clearly,further exploration of the mechanisms that link miR-23 and laminB1 to oligodendrocyte maturation and myelin maintenance mightprovide insights into new therapeutics for ADLD and otherdemyelinating disorders like multiple sclerosis.

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METHODSPlasmid constructionsThe cDNA encoding mouse Lmnb1 was amplified by polymerasechain reaction (PCR) from mouse brain total cDNA and wassubcloned into pCMV tag2 (Stratagene), pEGFP-C3 (Clontech)and pSicoR (Ventura et al., 2004). The 3�UTR from mouse Lmnb1was cloned into the pRL-TK vector (Promega) and mutations wereintroduced into the wild-type Lmnb1 3�UTR by PCR. microRNAprecursors with a 50-80 base pair flanking sequence on each sitewere amplified by PCR from mouse genomic DNA (Promega) andthen inserted into the pSuper (OligoEngine) and the pSicoRlentiviral vectors. All constructs used in this study were verifiedby sequencing (Genomics Core Facility, UCSF). GFP fusionlamin B1 (Daigle et al., 2001), lamin A (Ostlund et al., 2001) andlamin B2 constructs (BC 006551) (Open BioSystems) were madein pEGFP-C3 and GFP-GST vectors containing NLS, or NLS andNES (Walther et al., 2003). Anti-miR analysis of microRNAknockdown was performed using single-stranded RNAoligonucleotides designed to inhibit miR-23a and miR-23b(Dharmacon).

Cell culture and transfectionHEK293, C17.2 and N20.1 cells were grown as described previously(Snyder et al., 1992; Verity et al., 1993). The mouse fibroblasts wereisolated from adult LMNB1-overexpressing transgenic mice(unpublished data) following standard procedures (Xu et al., 2007).Primary glial cultures were performed using standard methods(McCarthy and de Vellis, 1980; Armstrong et al., 1992; Dugas etal., 2006). Plasmid or single-stranded RNA transfection wasperformed by using FuGene HD (Roche) or nucleofectorelectroporation with the Amaxa system (Amaxa Biosystems).

Virus generation and infectionLentiviruses were generated essentially as described previously(Ventura et al., 2004). Lentiviral vectors and each packaging vectorwere co-transfected into HEK293T cells. Supernatants werecollected 36-48 hours after transfection and were passed througha 0.45 μm filter. The viral supernatant was centrifuged at 100,000 gfor 1.5 hours. The viral pellet was resuspended in PBS andincubated overnight at 37°C with the cultured primary cells. Twoto four rounds of infection were performed 24 hours apart.

Luciferase reporter assayLuciferase and β-galactosidase activities in the cell extracts wereassayed 48 hours after transfection as described previously (Zhaoet al., 2005). pGL3-promoter or pSV-βGAL expression constructswere co-transfected to normalize for transfection efficiency.NeuroD-Luc, MBP-Luc, PLP-Luc, MOG-Luc (PCR cloning from–657~+80) or GFAP-Luc were used in this study.

MicroRNA northern blotting and quantitative RT-PCRRNA was isolated from cells and mouse brain using Trizol(Invitrogen) and microRNA northern blotting was performed asdescribed previously (Zhao et al., 2005). Quantitative PCR wasperformed using a RotorGene RG3000 real-time PCR system(Corbett Research) with the SYBR-green-containing PCR kit(BioRad). All PCR fragments were sequenced to validate thespecificity.

Immunohistochemistry, immunocytochemistry and western blotanalysisMice were perfused with 4% paraformaldehyde and the brains werepostfixed overnight at 4°C. Cryosections (20 μm) werepermeabilized with 0.3% Triton X-100 in PBS and then blockedwith 20% normal goat serum. Cells grown on coverslips were fixedin methanol at –20°C or 4% paraformaldehyde in PBS at roomtemperature. Cells were then permeabilized with 0.1% Triton X-100 in PBS followed by blocking with 5% nonfat dry milk. Thesecells or brain sections were subjected to primary antibody followedby Cy2- or Cy3-conjugated antibodies. Photographs were taken ona Zeiss Pascal confocal microscope with a 63� oil-immersionobjective.

Cell lysates were loaded onto 10% SDS-PAGE gels and were thentransferred onto PVDF membrane (Bio-Rad). Blots were blockedwith 5% nonfat dry milk followed by incubation with adequateprimary antibody in 2.5% nonfat dry milk. HRP-conjugatedsecondary antibody was then applied to the blots and bands werevisualized using an ECL chemiluminescence kit (Amersham) andautoradiograph (Pierce X-OMAT film).

Sholl analysis of oligodendrocytesNIH ImageJ software with Sholl analysis plugin was performed inoligodendrocytes following MBP immunocytochemistry. Tracedcells were analyzed by centering nested spheres on the cell bodywith each spheres spaced 10 μm apart (from 10 to 100 μm). Thecomplexity of oligodendrocyte morphology was presented bycounting how many times the branches intersect with thecircumference of these circles.

Statistical analysisData were presented as mean±s.e.m. and data comparison wasundertaken using the Student’s t-test or one-way ANOVA withNewman-Keuls post hoc test. The significant difference was set atP<0.05 unless otherwise stated.

PrimersThe primers for quantitative real-time PCR were as follows:Lmnb1 F: 5�-CAGGAATTGGAGGACATGCT-3� and R: 5�-GAAGGGCTTGGAGAGAGCTT-3� (40 cycles of 95°C for 10seconds, 59°C for 10 seconds, 72°C for 20 seconds; detectiontemperature at 82°C).GAPDH F: 5�-AACTTTGGCATTGTGGAAGG-3� and R: 5�-ACACATTGGGGGTAGGAACA-3� (40 cycles of 95°C for 10seconds, 60°C for 10 seconds, 72°C for 15 seconds; detectiontemperature at 82°C).

Oligodeoxynucleotides used as northern probes were as follows:miR-23a, 5�-GGAAATCCCTGGCAATGTGAT-3�; miR-23b, 5�-GGTAATCCCTGGCAATGTGAT-3�; U6, 5�-GCAGGGGCCAT -GCTAATCTTCTCTGTATCG-3�.

AntibodiesMonoclonal or polyclonal antibodies used inimmunohistochemistry and immunocytochemistry were as follows:CNP at a dilution of 1:250 (Abcam), GalC at 1:200 (Chemicon),GFAP at 1:500 (Chemicon), GFP at 1:1000 (Abcam), HP1β at 1:500(Chemicon), trimethyl-histone H3 (Lys4) at 1:250 (Upstate), tri-methyl-histone H3 (Lys9) at 1:250 (Upstate), Lamin B1 at 1:500

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(Abcam), LAP2 at 1:1000 (BD bioscience), LBR at 1:1000 (Dregeret al., 2002), MAG at 1:250 (Chemicon), MBP at 1:500 (Chemicon),NeuN at 1:250 (Chemicon), NG2 at 1:400 (Chemicon) and PLP at1:100 (Abcam).

Monoclonal or polyclonal antibodies used in western blottingwere as follows: CNP at a dilution of 1:1000 (Abcam), Flag M2 at1:5000 (Sigma), GAPDH at 1:5000 (Chemicon), GFAP at 1:3000(Chemicon), GFP at 1:5000 (Abcam), HDAC1 at 1:5000 (ABR),LAP2 at 1:1000 (BD bioscience), Lamin B1 at 1:1000 (Abcam), MBPat 1:1000 (Santa Cruz), LBR at 1:5000 (Dr Harald Herrmann), MAGat 1:500 (Zymed), NeuN at 1:1000 (Chemicon) and Nup153 at1:1000 (Abcam).

ACKNOWLEDGEMENTSWe thank E. Synder for the C17.2 cell line; A. Campagnoni for the N20.1 cell line; H.Worman for the lamin A cDNA construct; J. Ellenberg for the GFP-lamin B1(human) construct; M.-J. Tsai for the NeuroD-luciferase construct; R. Miskimins forthe MBP-luciferase construct; S. Goebbels and K. Nave for the PLP-luciferaseconstruct; I. Rozovsky for the GFAP-luciferase construct; E. Atlas and R. Haché forthe NLS-GFP and NLS-GFP-NES constructs; H. Herrmann for the LBR antibody; K.Luo for the MAN1 antibody; Q. Padiath for protein extracts of ADLD brains; D.Srivastava for microRNA targeting predictions; and L. Ptáček and S. Pleasure for

careful reading of the manuscript. We thank members of the Fu and Ptáček labsfor discussions and technical assistance. We especially thank B. Barres for helpingus to implement the various primary culturing methods from his laboratory. Thiswork was supported, in part, by the Sandler Neurogenetics fund (to Y.-H.F.).

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSS.T.L. conceived, designed and performed the experiments; analyzed the data; andwrote the paper. Y.-H.F. conceived and designed the experiments, analyzed thedata, and wrote the paper.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/content/2/3-4/178/suppl/DC1

Received 2 July 2008; Accepted 17 November 2008.

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TRANSLATIONAL IMPACT

Clinical issueDemyelination of the central nervous system (CNS) impairs nervous systemfunction and can create deficits in cognition, sensory perception andmovement, depending on the affected brain regions. CNS demyelination canresult from a number of genetically characterized, hereditary, childhood- andadult-onset leukodystrophies, the most common disorder being multiplesclerosis (MS).

Little is known about the morphological and molecular changes involved inmyelin sheath formation and maintenance. Studies of genetic forms ofhypomyelinating and demyelinating leukodystrophies provide uniqueopportunities to identify the important factors for myelin synthesis,maintenance and (potentially) regeneration. Adult-onset autosomal dominantleukodystrophy (ADLD) is one example where an unexpected structuralprotein, lamin B1, was found to be important for maintaining normalfunctional myelin. Lamins are major components of the nuclear envelope thatinfluence transcription factor localization. This study investigates howoverexpression of lamin B1 contributes to demyelination in the CNS.

ResultsIn this paper, the authors use a combination of established cell lines andprimary cell culture methods to study the role of lamin B1. Aberrant innernuclear membrane protein localization and nucleo-cytoplasmic traffickingprovide evidence of disrupted nuclear membrane functions. Lamin B1expression in cells was shown to be crucial for normal oligodendrocytedevelopment and differentiation, and oligodendrocyte development wasinhibited with lamin B1 overexpression. Furthermore, they demonstrated thatthe microRNA miR-23 represses lamin B1 protein levels, contributing to thetight regulation of lamin B throughout development and in adults.

Implications and future directionsThe results demonstrate that appropriate regulation of lamin B1 inoligodendrocyte precursor cells and in mature oligodendrocytes is crucial fornormal myelin maintenance. This suggests that therapeutic interventions thatreduce lamin B1 might be a potential treatment for ADLD patients. As themolecular mechanisms of myelin synthesis and maintenance are furtherdefined, proteins and microRNAs that are important for these processes can beinvestigated as potential drug targets for the prevention of myelindegeneration and/or to assist in myelin maintenance.

doi:10.1242/dmm.002576

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RESEARCH ARTICLE Lamin B1 and miR-23 in myelination

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miR-23 regulation of lamin B1 is crucial for ... · Disease Models & Mechanisms 179 Lamin B1 and miR-23in myelination RESEARCH ARTICLE uncover a novel function for a nuclear structural