Juxtanodin: An oligodendroglial that promotes cellular arborization and 2؅,3؅-cyclic nucleotide-3؅-phosphodiesterase trafficking

Bin Zhang*, Qiong Cao*, Anchen Guo*, Haiying Chu*, Yee Gek Chan*, Jan Paul Buschdorf†, Boon Chuan Low†, Eng Ang Ling*, and Fengyi Liang*‡

*Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 117597; and †Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543

Edited by Edward G. Jones, University of California, Davis, CA, and approved June 16, 2005 (received for review February 4, 2005) In the process of screening cell-type-specific genes, we identified databases revealed another cDNA sequence encoding a trun- juxtanodin (JN), an oligodendroglial protein featuring a putative cated JN of 268 residues (JN268). C-terminal -binding domain. At the cellular level, JN in the rat CNS colocalized with 2؅,3؅-cyclic nucleotide-3؅-phosphodiesterase Generation of Fusion and JN Antibody. The DNA fragments (CNPase), a cytoskeleton-related oligodendroglial protein. In the encoding residues 1–170 and 1–282 of deduced JN were cloned myelin sheath, JN was found mainly in the abaxon and the lateral into pET41a (Novagen) to produce GST-JN170 and His-tagged few terminal loops. Its apposition to the myelinated axon, through JN. GST and GST-JN170 were purified by using Bulk and the latter, defined an axonal subregion, herewith termed juxta- RediPack GST Purification modules (Amersham Pharmacia, node, at the Ranvier node–paranode junction. During forebrain Piscataway, NJ). Polyclonal antibody against GST-JN170 was ontogenesis, JN expression paralleled that of MBPs but lagged developed in New Zealand White rabbits and affinity purified behind CNPase. Juxtanodin transfection promoted arborization of with GST-JN170-Sepharose 4B. cultured OLN-93 cells and augmented endogenous CNPase expres- sion and transport to the process arbors of cultured primary Mammalian Expression Plasmids and Transfection of Cell Culture. The oligodendrocyte precursors. These results reveal JN as a cytoskel- spontaneously transformed, microfilament-containing OLN-93 eton-related oligodendroglial protein that delineates the juxtan- cells (5) were cultured at 37°C in DMEM supplemented with ode and might serve oligodendrocyte motility, differentiation, or 10% FCS. Primary oligodendrocyte precursor (OLP) cells were myelin–axon signaling. Functionally, JN may be involved in CNS prepared from the optic nerves of 5- to 7-day-old rats (6, 7) in myelination and͞or specialization of the node of Ranvier. the facilities at the Department of Clinical Research, Singapore General Hospital (Singapore). myelin–axon interaction ͉ oligodendrocyte ͉ node of Ranvier ͉ cytoskeleton To elucidate the functional roles of JN and its putative C-terminal actin-binding domain (as reported by National Cen- ligodendroglia are highly specialized myelin-forming cells ter for Biotechnology Information reverse-position-specific Oof the CNS. Adequate myelination serves to insulate and BLAST), the nucleotide sequences encoding JN (282 amino acid accelerate the propagation of action potentials. Abnormalities in residues), JN268, and JN residues 1–247, 1–141, and 101–282 were Ј myelin formation͞maintenance may underlie diverse neurolog- subcloned into pXJ40 (8) with a FLAG tag at the 5 end of the ical disorders, ranging from multiple sclerosis to schizophrenia inserts, generating the constructs for FLAG-tagged JN, JN268, (1, 2). Matched with highly specialized functions and unique JN247,JN141, and JNc, respectively. Empty pXJ40 and pXJ40- architecture, various oligodendrocyte͞myelin-selective mole- BNIP-S served as controls (9). Cultured cells were transfected cules, such as myelin basic protein (MBP), myelin-associated with the recombinant plasmids by using GIBCO Lipofectamine glycoprotein, and 2Ј,3Ј-cyclic nucleotide-3Ј-phosphodiesterase 2000 (Invitrogen). Forty-eight hours later, the cells were solu- (CNPase), have been isolated and characterized (1, 3). It is likely bilized for Western blotting or fixed with 3% paraformaldehyde that many more are yet to be revealed. The identification and for immunocytochemistry. characterization of such molecules will probably help elucidate molecular mechanisms of myelination and dys- or demyelinating Immunocytochemistry, Electron Microscopy, and Multiple Labeling. Adult rats (n ϭ 18, body weight Ϸ200 g) were deeply anesthe- diseases. ͞ The unique architecture of the myelin sheath entails special- tized with Nembutal (100 mg kg of body weight, i.p.) and NEUROSCIENCE ized, yet elusive, cytoskeletal mechanisms of myelin-forming transcardially perfused with saline, followed by 3% paraformal- cells. Here, we report the molecular features, cellular expression, dehyde (plus 0.1% glutaraldehyde for immunoelectron micros- and functional roles of juxtanodin (JN), a previously unidentified copy) in 0.1 M phosphate buffer (pH 7.4). The brain and spinal cytoskeleton-related oligodendrocyte-specific protein. cord were dissected, postfixed, and sectioned with a cryostat (for light microscopy) or a vibratome (for electron microscopy). All Materials and Methods procedures involving experimental animals were approved by Identification and Characterization of the mRNA. Cell-type-specific the Ethics Committee at the National University of Singapore. CNS genes were sought out by in situ hybridization histochem- istry (ISH). Briefly, cDNA clones (n ϭ 1,500) from a rat brain This paper was submitted directly (Track II) to the PNAS office. cDNA library (Invitrogen) were sequenced and compared by Abbreviations: CNPase, 2Ј,3Ј-cyclic nucleotide-3Ј-phosphodiesterase; ERM, –– BLAST searches against National Center for Biotechnology In- ; FERM, band four-point-one ERM homology; ISH, in situ hybridization histochem- formation nucleotide and protein nonredundant databases (4). istry; JN, juxtanodin; MBP, myelin basic protein; OLP, primary oligodendrocyte precursor; For the unannotated cDNA sequences (n ϭ 274), expression of PD, postnatal day. the mRNAs in the CNS was mapped by ISH using digoxigenin- Data deposition: The sequence reported in this paper has been deposited in the GenBank labeled riboprobes. A 3.6-kb cDNA clone with a predicted ORF database (accession no. DQ119821). encoding 282 amino acid residues was thereby identified, and the ‡To whom correspondence should be addressed. E-mail: [email protected]. gene was subsequently named juxtanodin (JN). A search of EST © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500952102 PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11527–11532 Downloaded by guest on September 29, 2021 The following antibodies were used (mouse monoclonal an- 282). BLAST searches against National Center for Biotechnology tibody from Sigma, unless otherwise noted): anti-JN (1:100, Information nonredundant nucleotide and protein databases rabbit polyclonal antibody, in-house produced), anti-CNPase yielded no positive hits, except for an unnamed mRNA sequence (1:500, Chemicon), anti-FLAG (1:200), anti-glial fibrillary acidic predicted by automated genome computation (GenBank acces- protein (1:1,000, Chemicon), anti-MBP (1:1,000, goat polyclonal sion no. NM࿝001008311, 100% identity with JN), a deduced antibody, Santa Cruz Biotechnology), anti-neurofilament 200 human KIAA1189 protein (284 amino acid residues, 58% amino (1:1,000), anti-OX42 (1:50, Harlan Sera-lab, Sussex, U.K.), acid identity with JN), and an unnamed mouse protein inferred anti-pan sodium channel (NavP, 1:200), and anti-potassium from a cDNA sequence (accession no. NP࿝084248, 235 amino channel Kv1.2 (1:300, Upstate Biotechnology, Lake Placid, NY). acid residues, 64% amino acid identity with JN). EST databases In vitro transcription of digoxigenin-labeled riboprobes, ISH presented a cDNA clone encoding a truncated JN consisting of (probe concentration, 0.2 ␮g͞ml), immunofluorescence (IF), 268 amino acid residues (JN268; Fig. 1A). immunoperoxidase (avidin-biotinylated peroxidase complex In comparison with characterized proteins, the JN C terminus method), and immunoelectron microscopy followed protocols showed homology to the actin-binding domain of the ezrin- described in refs. 10 and 11. For simultaneous IF double͞triple radixin-moesin (ERM) proteins. Fig. 1A compares JN and JN268 labeling, bound primary antibodies were revealed by appropriate with moesin (NCBI accession no. secondary antibodies conjugated to either Alexa Fluor 568 or AAB61666). The boxed residues denote the homologous region Alexa Fluor 488 (1:400, Invitrogen). For sequential double- among the three. Unlike the ERM proteins, however, JN labeling, IF signals for the first antigen were documented before exhibited no detectable plasma-membrane-binding FERM immunoperoxidase for the second antigen and Luxol fast blue (band four-point-one ERM homology) domain. Instead, a scan counterstaining (0.1% in 95% ethanol, overnight at 50°C) for the of the Prosite database of protein families and domain patterns visualization of immunonegative cell arbors. Unexpected cross (12) reported four potential N-myristoylation sites near the JN reactivity in double͞triple labeling could be ruled out, based on N terminus (at residues 10–15, 22–27, 43–48, and 61–66; un- control experiments in which one of the primary antibodies was derlined by dots in Fig. 1A). omitted. On Western blots of adult rat brain lysate, anti-JN antibody, but not preimmune or GST-JN170 preabsorbed serum, detected Immunoprecipitation and Northern and Western Blot Analyses. Rats a 41-kDa major band that occasionally appeared as a closely were killed by the injection of Nembutal (100–150 mg͞kg of body spaced doublet (double arrows in Fig. 1D, lane 1). A band at the weight, i.p.), and the tissues were dissected and homogenized. same position was also obtained from immunocomplexes of rat For immunoprecipitation, solubilized proteins were precleared brain lysate precipitated with the JN antibody (Fig. 1D, lane 2) by protein A-agarose (Amersham Pharmacia) and incubated but not with the preimmune rabbit serum (Fig. 1D, lane 3). Actin with 5 ␮g of JN antibody or rabbit IgG (control) followed by 25 was coimmunoprecipitated with JN by the JN antibody (Fig. 1D, ␮l of protein A-agarose (overnight at 4°C). Immunoprecipitated lane 9). FLAG-tagged JN (by transfected 293T cells) or His- proteins were separated by SDS͞PAGE. For Northern blot, tagged JN (by transformed BL21 Escherichia coli), as detected by poly(A)ϩ RNAs from various tissues were separated on 1% JN antibody, migrated to Ϸ43 kDa on Western blot (Fig. 1D, agarose͞formaldehyde gel and transferred onto charged nylon lanes 6 and 8). The position of the former was confirmed by the membrane (PerkinElmer). Overnight hybridization with digoxi- anti-FLAG antibody (lane 5). No FLAG or JN signal was genin-labeled JN riboprobe (15 ng͞ml) was followed by washes revealed on the blots of cells transfected with empty pXJ40 and detection with alkaline phosphatase-labeled anti- vector (Fig. 1D, lanes 4 and 7). digoxigenin antibody and CDP-Star reagent (Roche, Basel, The above immunoblotting results showed a discrepancy Switzerland). For Western blot, samples were separated on between the calculated molecular mass (32.2 kDa) and the actual SDS͞PAGE and transferred to PolyScreen poly(vinylidene di- migration of JN on SDS͞PAGE. Further tests attributed this fluoride) membrane (PerkinElmer). The primary antibody la- atypical migration of JN largely to its residues 101–247. As shown beling was detected with alkaline-phosphatase-conjugated sec- in Fig. 1C (lanes 2–5), in vitro expression of FLAG-tagged JN, ondary antibodies and CDP-Star reagent. JN247,JN141, and JNc yielded products that, on Western blots, measured heavier than predicted by 30%, 41%, 16%, and 44%, Data Analyses. Immunofluorescent and immunoelectron micro- respectively. scopic preparations were analyzed by using a laser scanning confocal microscope (Fluoview FV500, Olympus, Tokyo) and an Expression Profile of JN in the Developing Forebrain. In the devel- electron microscope (Philips EM208S, FEI, Eindhoven, The oping forebrain, JN immunoreactivity emerged at about the Netherlands), respectively. Molecular masses of protein or same time as major MBP isoforms but lagged behind CNPase mRNA bands on the blots were estimated by using the program expression by Ϸ2–3 days. As shown in Fig. 1 (E and F), JN signal GENETOOLS (Syngene, Cambridge, U.K.). Integrated OD was was undetected at postnatal day (PD) 0 through PD 7, remained calculated by multiplying the mean OD by the area (mm2)ofthe very weak at PD 10, rapidly increased for the period PD 14–PD positive band above background by using the program IMAGEJ 21, and approximated the adult level by PD 28. Similarly, faint 1.33U (National Institutes of Health, Bethesda). OD was defined MBP signals appeared at PD 10. The 17-kDa MBP gradually on 8-bit black͞white images with gray level 255 (white) as 0 OD diminished after peaking at PD 14 (indicated by a filled arrow- and gray level 0 (black) as 2.708 OD. head in Fig. 1F). Some other MBP isoforms quickly reached plateau expression by PD 14–PD 21 and stayed high thereafter Results (indicated by open arrowheads in Fig. 1F). Significant CNPase Molecular Features of JN. On multitissue Northern blots, the JN signal, on the other hand, was discerned as early as PD 7 and cRNA probe detected two major transcripts of 4.0 kb and 1.9 kb increased gradually to about the adult level at PD 28. in rat brain poly(A)ϩ RNA. No hybridization signal was found in poly(A)ϩ RNAs of the other seven tissues examined (Fig. 1B). Cellular Expression of JN in the CNS. By using ISH, JN mRNA was The 3.6-kb JN cDNA clone had a predicted ORF of 846 bp and detected in the perikarya of glia-like cells throughout the CNS. a5Ј untranslated region of 80 bp with two in-frame stop codons. Positive cell bodies appeared generally small and scattered in the The deduced peptide JN consisted of 282 amino acid residues gray matter, and they were more numerous, morphologically (predicted Mr ϭ 32,226; pI ϭ 4.64) with high contents of more variable, and often queued along the direction of axon glutamic acids (14.89%, 42 of 282) and serines (9.57%, 27 of passage in the white matter (Fig. 2). Overall, the spinal cord

11528 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500952102 Zhang et al. Downloaded by guest on September 29, 2021 Fig. 1. JN’s molecular features and expression profile in the postnatal forebrains. (A) Alignment and comparison of JN and JN268 with rat moesin, a typical ERM protein. Boxed sequences at the C termini denote the putative actin-binding domain. Identical residues are shaded, low-complexity regions are underlined, and potential N-myristoylation sites (reported by scanning Prosite patterns) are underlined by dots. (B) Multitissue Northern blot hybridized with JN-specific riboprobe. Two JN transcripts were revealed in the brain but not in the other seven tissues (SkM, skeletal muscle). (Bottom) The strip shows the SYBR gold staining of 28S rRNA on the nylon membrane. (C) Western blots, comparing the mobility of native brain JN (lane 1) with FLAG-tagged JN, JN247,JN141, and JNc (lanes 2–5). Framed schematic diagrams at the top represent the respective proteins. The filled triangles on the left sides of the diagrams stand for the N-terminal FLAG tag. The filled ellipses on the right sides of the diagrams denote the putative C-terminal actin-binding domain of JN. Differences (Diff) between predicted (Pred) molecular masses and actual migrations (Mig) of the proteins are shown below the blots. (D) Western blots, showing molecular analyses of JN. Labels below the blots indicate antibodies used for detections. The blot samples are from brain (lane 1), immunoprecipitates of rat brain lysate by the anti-JN antibody (lanes 2 and 9) or by preimmune rabbit serum (lane 3), 293T cells transfected by empty pXJ40 plasmid (lanes 4 and 7) or by FLAG-tagged JN (lanes 5 and 6), and His-tagged JN expressed by E. coli (lane 8). (E) Line graphs, showing relative levels of JN (Upper) or CNPase (Lower) immunoreactivities in the PD 0, 3, 7, 10, 14, 21, 28, and 60 rat forebrains. See Materials and Methods for quantification of positive bands on Western blots. (F) Western blots, comparing expression of CNPase, JN, MBPs, and actin in the PD 0, 3, 7, 10, 14, 21, 28, and 60 rat forebrains. Each lane was loaded with 20 ␮g of solubilized proteins.

showed the highest expression levels and the densest JN-positive section of spinal white matter. Between the Kv1.2-positive signals, followed by the brainstem, cerebellum, thalamus, and juxtaparanodes of adjacent myelin sheaths, two narrow bands of hypothalamus. In the cerebral and cerebellar cortices, positive intense JN reactivity were found adjoining a JN-negative zone. cells were scarce superficially but increased markedly toward Triple staining of JN, Kv1.2, and NavP established the latter zone deeper layers (Fig. 2). No significant JN labeling was detected in as the node of Ranvier (with NavP clusters) and ascertained its spinal dorsal roots (data not shown). close relation with the flanking JN bands (Fig. 3H). Distribution of JN immunoreactive cell bodies in the CNS Immunoelectron microscopy confirmed the presence of JN in agreed with that of the mRNA. Additionally, immunopositive oligodendroglial perikarya and processes (Fig. 4A). Although Ј signals along axons͞fiber tracts were observed throughout the the immunoreactivities appeared clustered, JN s relation to CNS, especially in the white matter (Fig. 3 A–H). On cross specific organelles or the cytoplasmic membrane could not be sections of fiber tracts, such as those in the spinal white matter, established. Of the myelin sheath, strong signals were observed NEUROSCIENCE JN-immunoreactive profiles were seen around the neurofila- in the outermost cytoplasm-containing layer (abaxon or outer loop͞‘‘tongue’’). To a lesser extent, JN was also present in the ment-200-positive axons and, often, separated from the latter by cytoplasm-containing inner layer lining the compact myelin a negative zone (Fig. 3E). At the cellular level, JN colocalized sheath (adaxon or inner loop͞tongue; Fig. 4 A and B). Of the with CNPase in all CNS regions examined. Subcellularly, JN terminal loops, only those lateral few (mostly numbers 1–3) distribution overlapped that of CNPase substantially but not adjoining the node of Ranvier showed clear deposits of JN completely. On cross section of fiber tracts of cervical spinal immunoreactivity that, on occasion, could be seen associated white matter, for example, CNPase often labeled two concentric with the loop membrane or submembranous layer (Fig. 4 C and rings, corresponding, probably, to the abaxonal and adaxonal D). Medial terminal loops further away from the node appeared layers of the myelin sheath. JN coexisted with CNPase mostly in to be devoid of significant JN immunoreactivity, as were the the abaxonal (outer) ring and, at times, a small part of the compact lamellae of the myelin sheath. adaxonal (inner) ring (Fig. 3C). No significant JN colocalization Based on its distribution and site of apposition with the was found with glial fibrillary acidic protein in astrocytes, OX42 myelinated axon, we named this protein juxtanodin and propose in microglia, or neurofilament 200 in neurons (Fig. 3 D–F). a juxtanode domain of myelinated axon at the Ranvier node– Confocal microscopy and multiple immunofluorescence fur- paranode junction (Fig. 4E, Jn). Fig. 4 E and F compares the ther revealed JN-enriched narrow bands or dots flanking the proposed with the conventional segmental divisions of myelin- nodes of Ranvier. Fig. 3G shows such examples on a longitudinal ated axon and myelin sheath.

Zhang et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11529 Downloaded by guest on September 29, 2021 Fig. 2. JN mRNA expression in the CNS, as revealed by ISH in the deep layers of frontal cerebral cortex (A), ventral midbrain region including the substantia nigra (B), cerebellar cortex (C), and cervical spinal cord (D). 5 and 6, layers V and Fig. 3. Confocal photomicrographs showing JN distribution in the CNS. (A) VI of frontal cerebral cortex; GM, gray matter; SN, substantia nigra; WM, white JN immunofluorescence in the cerebellar cortex. Note the JN-positive oligo- matter. (Scale bar: 0.2 mm.) dendrocyte at the border between molecular (Mol) and Purkinje cell (Pur) layers. (B–F) Double immunofluorescence of JN with CNPase (B and C), GFAP (D), neurofilament 200 (E), or OX42 (F) in layers III and IV of frontal cortex (B), JN Promotes Arborization of Cultured Oligodendrocytes. OLN-93 cross sections (C, E, and F), or longitudinal section (D) of cervical spinal white cells, under normal culture conditions, showed no JN immuno- matter. (G) Double immunofluorescence of JN and Kv1.2 on longitudinal reactivity. At low to medium densities, the majority were bipolar section of spinal white matter. Arrows point to the presumptive positions of in morphology, although some displayed more than two primary the nodes of Ranvier. (H) Triple staining of JN (red), Kv1.2 (green), and NavP cellular processes. In general, the cell bodies and processes were (green) on a longitudinal section of spinal white matter. Arrows point to examples of the NavP clusters at the nodes of Ranvier. (Inset) An example from smooth on the surface and gave rise to only a few branches. ͞ At 48 h after being transfected, Ϸ5–10% of cells in the culture another microscopic field. Note the JN-enriched bands dots flanking the node of Ranvier and between the strongly Kv1.2-positive juxtaparanodes. In all expressed the extrinsic protein, as determined by immunocyto- panels, single-labeling would appear red (JN) or green (others). Yellow indi- chemistry. In comparison with neighboring untransfected cells in cates double-labeling. Open arrowheads denote cell bodies of oligodendro- the same culture, JN- and JNc-transfected OLN-93 cells exhib- cytes. (Scale bars: 10 ␮m.) ited not only longer and more numerous primary processes but also significantly finer process arbors with many filopodium-like prickles from the processes or directly from the cell bodies (Fig. Discussion 5 A and B). Some transfected cells also gave rise to lamellopo- This study identified JN as a cytoskeleton-related oligodendro- dium-like protrusions. JN268 transfection produced similar but glial protein. Of CNS myelinated axons, JN distribution in the much weaker effects. Transfection by JN247,JN141, empty pXJ40 node-abutting lateral terminal loops of myelin sheath morpho- vector, or the JN-unrelated BNIP-S (9), however, did not induce logically defined a juxtanode region between the paranode and clear morphological change in OLN-93 processes (Fig. 5). the node of Ranvier. JN emergence during CNS ontogenesis paralleled that of major MBPs and coincided with the onset of JN Promotes CNPase Trafficking to the Site of Action in Primary OLP myelination. JN transfection induced extensive process out- Cells. Primary OLP cells from 5- to 7-day-old rat optic nerve did growth and filopodium-like-protrusion formation in cultured not yet exhibit significant intrinsic JN immunoreactivity at the OLN-93 cells and enhanced endogenous CNPase expression and stage of 7–9 days in culture. JN transfection elicited more transport to the site of action at the process arbors of primary extensive arborization and branching of the cells, in comparison OLPs. with the untransfected cells and cells transfected with JN247 or JN141 (Fig. 6 A, C, and E). Moreover, JN transfection up- JN: An Oligodendroglia Marker of a Previously Unidentified Protein regulated CNPase expression and facilitated CNPase transport Family. As evidenced by our double-labeling results, JN was a to the process arbors. As a result, the transfected OLPs showed specific marker for oligodendroglia in the CNS. In contrast to markedly higher levels of CNPase immunoreactivity, especially CNPase, neither JN mRNA nor protein was detected in the in their processes (Fig. 6 A and B). The untransfected surround- Schwann cells of spinal dorsal roots, suggesting its absence in the ing OLPs exhibited CNPase signals, mainly in the perikarya (Fig. peripheral nervous system and, therefore, higher expression 6B). Similar effects, although less prominent, were observed in specificity. cells transfected with JNc (data not shown). Transfection with Although exhibiting an ERM-like C-terminal actin-binding JN247 and JN141 produced increasingly weaker, but still observ- domain, JN lacked the N-terminal membrane-binding FERM able, facilitatory effects on CNPase expression and trafficking domain found in all known ERM proteins (13). Hence, it might (Fig. 6 C–F). not belong to the ERM protein family. It is unknown whether

11530 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500952102 Zhang et al. Downloaded by guest on September 29, 2021 Fig. 5. Effects of JN expression on OLN-93 arborization, as revealed by immunoperoxidase by using the anti-JN (A–E) or anti-FLAG (F and G) antibody. The preparations were lightly counterstained with cresyl violet to visualize untransfected cells. JN-transfected OLN-93 cells exhibited longer and more numerous processes and finer process arbors (A). JNc transfection produced similar but weaker effects (B). Fewer morphological changes were seen in cells transfected with JN268 (C). No enhanced process arborization was seen when cells were transfected with JN247 (D), JN141 (E), BNIP-S (F), or empty pXJ40 vector (G). (H) Summary of the transfection results in cultured OLN-93 cells. In schematic diagrams representing the fusion proteins, the filled triangles on the left stand for the N-terminal FLAG tag, and the filled ellipses on the right denote the putative C-terminal actin-binding domain of JN. (Scale bar: 20 ␮m.) Fig. 4. Electron micrographs, showing JN immunoreactivity in oligodendro- cyte and myelin sheaths. (A and B) Electron micrographs of the cerebellum, showing JN in the cytoplasm of an oligodendrocyte (A), in the abaxons (open JN C terminus resembled the well characterized actin-binding arrows), and, occasionally, in the adaxons (open arrowheads) but not in the domain of ERM proteins. (iii) JN expression promoted arboriza- compact layers of the myelin sheath (B). (C and D) On longitudinal sections of myelinated axons in cervical spinal cord (C) or corpus callosum (D), JN immu- tion and CNPase trafficking to the process arbors of cultured noreactivity was observed in the lateral few terminal loops (filled arrows) primary OLPs. (iv) Actin was coimmunoprecipitated with JN. abutting the node of Ranvier. The axonal segment surrounded by the JN- The physiological functions of CNPase have been attributed to positive lateral terminal loops is herewith defined as the juxtanode. (E and F) its roles in regulating tubulin polymerization͞distribution (17) Schematic diagrams comparing the suggested (E) with the conventional (F) and in myelin–axon interaction. CNPase also cross-links with domains of myelinated axon. The internode is not shown. a, axon; Jn, juxta- actin (18, 19). Like JN, CNPase is found mainly in the cytoplasm- node; JP, juxtaparanode; N, node of Ranvier; OL, oligodendrocyte; PN, para- containing noncompact regions of the myelin sheath (20, 21). In node. (Scale bars: 1.0 ␮minA, 0.5 ␮minB, and 0.3 ␮minC and D.) CNPase1-knockout mice, axons undergo swelling and degener- ation without significant abnormality of the myelin sheath (22). and how the potential N-myristoylation sites near the JN N These findings suggest a CNPase role in axon support and terminus, as predicted by Prosite patterns, could serve a similar myelin–axon interaction through as yet unknown mechanisms. membrane-binding function. The exact relationship between JN and the cytoskeleton The existence of JN isoforms is supported by our Northern remains unclear. For the induction of oligodendroglial arboriza- blot results and by BLAST searches of EST cDNA databases. It is tion, the C-terminal ERM-like actin-binding domain of JN worth noting that JN268 exhibited diminished arborization- promoting capability, as compared with JN. Shortening the putative actin-binding domain in the former may also affect subcellular localization of the protein. The atypical mobility of CNS native JN on SDS͞PAGE was reproduced by in vitro expressed JN. It remains unclear how this NEUROSCIENCE unusual feature of the protein arose. Our data indicated a greater contribution by the middle fragment of JN. Of particular interest, in this regard, was the high content of glutamic acids in the fragment (18.49% by frequency), especially in the region of low complexity (residues 172–189).

JN, Actin Cytoskeleton, and Myelin Sheath. The unique architecture of the myelin sheath calls for dedicated oligodendroglial cy- toskeletal machinery in the CNS (14, 15). Tubulin, actin, and associated proteins have been well documented in oligodendro- cytes, but the molecular mechanisms underlying the motility of the oligodendroglia͞myelin sheath remain poorly understood Fig. 6. Photomicrographs, showing the effects of JN (A and B), JN247 (C and ͞ D), or JN141 transfection (E and F) on primary OLP process ramification (A, C, (16). The following evidence supports a role of JN in binding and E) and CNPase expression and transport to the process arbors (B, D, and F). regulating oligodendroglial cytoskeleton: (i) JN showed substan- The cells were sequentially processed for CNPase immunofluorescence (red in tial colocalization in the myelin sheath with CNPase, a known B, D, and F), JN immunoperoxidase (brownish black in A, C, and E), and Luxol cytoskeleton-related oligodendroglial protein (17–19). (ii) The fast blue counterstaining (blue in A, C, and E). (Scale bar: 50 ␮m.)

Zhang et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11531 Downloaded by guest on September 29, 2021 appeared to be directly involved. Our transfection results indi- tribution of certain marker molecules, such as CNPase and cated that the C-terminal 181 residues of JN were sufficient. In MBPs, and the formation of myelin sheath (1). Expression of JN particular, the last 35 residues of JN, in which resides the in the developing forebrain lagged behind that of CNPase, potential ERM-like actin-binding domain, seemed to be indis- paralleled those of major MBP isoforms, and coincided with the pensable. The ERM proteins function as conformationally reg- onset of myelination therein (1, 23). This order of expression- ulated membrane–cytoskeleton crosslinkers in actin-rich surface onset of CNPase, MBPs, and JN in vivo seemed to be preserved structures, such as microvilli, microspikes, and membrane ruf- in cultured primary OLPs, in which JN did not appear before at fles, by using their C-terminal F-actin-binding domain (the last least 10 days in culture (data not shown). Our immunohisto- 34 residues) and an N-terminal membrane-binding FERM do- chemical studies in the developing CNS also confirm this ob- main (13). Cellular process extension and filopodium formation servation (data not shown). In vitro, JN transfection not only are known actin-filament-based processes (14, 15). Together, promoted process arborization but also increased CNPase ex- these findings suggest that JN most likely exerted its effects on pression and transport to the process arbors of cultured OLN- cell motility and arborization by interacting with and͞or influ- 93͞primary OLP cells. These data suggest structural integration encing the actin cytoskeleton of oligodendroglia. of exogenous JN into the host-cell cytoskeleton, functional Apart from affecting actin cytoskeleton through the C- activity of the protein for intracellular trafficking͞cellular mo- terminal domain, JN is likely to also function by other mecha- tility, and coordinated maturational acceleration of cultured nisms. JNЈs promotion of CNPase expression and transport, for oligodendrocytes in response to the exogenous JN. Taken to- example, was increasingly diminished, but not totally abolished, gether, our results indicate an important role of JN in late-stage by truncation of the C-terminal 35 or even 141 amino acid oligodendroglia maturation, in myelin͞Ranvier node formation residues. Future studies will be needed to clarify the cytoskeletal during CNS development, and in the maintenance and plasticity or other molecular elements responsible for these JN effects on of related structures in the mature CNS. CNPase. In the peripheral nervous system (PNS), ERM proteins en- riched in the perinodal Schwann cell microvilli are strongly implicated in inducing Ranvier node formation (24, 25). In the JN and the Delineation of the Juxtanode. As revealed by confocal ϩ microcopy and JN immunoelectron microscopy, the conven- CNS, oligodendroglia are known to similarly direct nodal Na tional terminal (paranodal) loops of myelin sheath are not channel clustering but are devoid of ERM proteins (26). We homogeneous in their molecular composition. The JN-positive hypothesize that JN serves a CNS function similar to that of the lateral terminal loops neighboring the node of Ranvier clearly ERM proteins in the PNS, based on its putative ERM-like differ from those JN-negative medial terminal loops adjoining actin-binding domain, its influence on oligodendroglial-process ͞ the juxtaparanode. Accompanying this molecular heterogeneity, outgrowth branching to approach the putative target axons, and its selective positioning to the juxtanodal terminal loops next to functional disparity between the two is expected. Hence, the ͞ proposed distinction of juxtanode from the paranode may bear the node. It remains unknown to what proteins structures, if any, important functional implications. The former might, for exam- JN links the actin cytoskeleton and how these molecules relate ple, be more related to myelin–axon interaction or to induction͞ to those downstream on the myelin–axon signaling cascade to sustaining of the node of Ranvier, whereas the latter may be influence node formation. more involved in the formation͞maintenance of compact myelin lamellae. We thank Dr. B. L. Tang (National University of Sinapore) for providing antibodies, Dr. J. H. Tang for technical assistance, and Mr. Y. H. Ou Yang for reading the manuscript. This work was supported by Biomed- JN, Myelination, and Specialization of the Node of Ranvier. The ical Research Council Grant 01͞1͞21͞19͞179 (to F.L.) and National maturation of oligodendrocytes is characterized by process out- University of Singapore Academic Research Fund Grant R181-000-052- growth and refinement, the programmed appearance and redis- 112 (to F.L.).

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