Dnternexin, a Novel Neuronal Intermediate Filament Protein, Precedes the Low Molecular Weight Neurofilament Protein (NF-L) in the Developing Rat Brain
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The Journal of Neuroscience, August 1990, W(8): 2735-2748 dnternexin, a Novel Neuronal Intermediate Filament Protein, Precedes the Low Molecular Weight Neurofilament Protein (NF-L) in the Developing Rat Brain Michael P. Kaplan, Steven S. M. Chin, Karsten H. Fliegner, and Ronald K. H. Liem DeDartments of Patholoav and Anatomv and Cell Bioloav, Columbia University College of Physicians and Surgeons, New Yolk, New York 10032 -’ cr-lnternexin is a 66 kDa protein that copurifies with inter- rons in the central and peripheral nervous system. A fourth mediate filaments (IF) from rat spinal cord and optic nerve. neuronal intermediate filament protein has recently been de- This protein is axonally transported in rat optic nerve along scribed (Portier et al., 1984; Parysek and Goldman, 1987; Leon- with the neurofilament triplet proteins in slow component a. ard et al., 1988); termed “peripherin,” this 57 kDa protein is Polymerization in vitro and distribution in viva confirm that found primarily in processesof the peripheral nervous system. cr-internexin is a neuronal IF. We raised 2 highly specific While their exact functions remain unclear, neuronal IFS pre- monoclonal antibodies to a-internexin which were applied sumably play some structural role in the generation and/or to frozen rat brain sections and Western blots of cytoskeletal maintenance of the highly asymmetrical forms of neurons and extracts. These results indicate that cY-internexin is primarily their processes;for example, it has been suggestedthat neuronal an axonal protein found in most, if not all, neurons of the IFS may be arbiters of neuronal caliber (for review, seeLasek CNS. lmmunoreactive proteins of similar molecular weight et al., 1983). were found in cytoskeletal extracts of CNS tissue from sev- Like other IFS, such as glial fibrillary acidic protein (GFA) in eral additional species, including mouse and cow. While the astroglia, desmin in smooth musclecells, and vimentin in cells distribution of cr-internexin as given by immunocytochemical of mesenchymalorigin, the neuronal IF proteins each contain methods is similar to that of low molecular weight neurofila- a highly conserved a-helical domain, flanked by variable head ment protein (NF-L) in the adult, its distribution in the embryo and tail sequences(for review, see Steinert and Roop, 1988); is far more extensive. At embryonic day 16, when the expres- the rod-shaped,a-helical domains form the core of the filament, sion of NF-L is still limited to a relatively small number of with the variable end sequencesprojecting outward. The exten- cells and levels of expression are low, a-internexin is already sive carboxy-terminal end sequenceof NF-H is known to par- found at much higher levels and in cells not yet expressing ticipate in the formation of cross-bridgesbetween filaments (Hi- NF-L in detectable quantities. Similar results are found at rokawa et al., 1984). NF-L, but not NF-M or NF-H, is readily embryonic day 12. These data suggest that neuronal IF in able to self-polymerize in vitro at physiological pH (Liem and the developing nervous system contain a higher proportion Hutchison, 1982); inability of the other isolated subunits to of cu-internexin than their adult counterparts, and that expres- polymerize is probably due to ionic repulsion between their sion of cY-internexin precedes that of NF-L in many or most highly phosphorylated tail sequences,and it has recently been neurons of the developing brain. suggestedthat this repulsion between phosphate groups may play a role in maintaining the distance between filaments in vivo Mammalian neuronal intermediate filaments (IF) have long been (Hirokawa et al., 1984; Carden et al., 1987; Shaw, in press). describedin terms of a “neurofilament triplet” of low, medium, Developmental studiesusing biochemical and immunological and high molecular weight proteins, designatedNF-L, NF-M, methods have shown that neurogenesisis accompanied by and NF-H, respectively (Hoffman and Lasek, 1975; Liem et al., changesin neuronal IF composition. After an initial stageduring 1978). Insoluble at physiological pH, they polymerize in vivo to which vimentin may be expressed(Bignami et al., 1982; Co- form 10 nm IFS which traverse the length of the neuronal axo- chard and Paulin, 1984), coexpressionof NF-L and NF-M pre- plasm. Filaments composed of variable proportions of the 3 cedesthat of NF-H in developing rat brain (Shaw and Weber, neurofilament proteins can be detected in the axons (and, to a 1982; Carden et al., 1987) retina (Shaw and Weber, 1983), and lesserextent, the dendrites and perikarya) of most mature neu- optic nerve (Pachter and Liem, 1984). Neuronal IFS containing NF-L and NF-M can be detected as early as embryonic day 12 Received March 7, 1990; revised April 24, 1990; accepted April 25, 1990. in some regions, while NF-H may not be expresseduntil days We thank Dr. James Goldman and Dr. John Pintar for their expert advice on or weeksafter birth in some areas. Hippocampal neurons cul- developmental anatomy, Mr. David Weinstein and Mr. Ray Manson for photo- tured in vitro display a similar stepwisepattern of expression graphic work, and Ms. Kristy Brown for electron microscopy. This work was (Shaw et al., 1985). The significanceof this delayed appearance supported by Grants NS 15 182 (Jacob Javits Neuroscience Award) and EY03849. K.H.F. and S.S.M.C. are supported by a Medical Scientist Training Program Grant of the larger subunit is probably related to its putative role as at New York University School of Medicine. cross-linker between filaments and the stabilization of IF struc- Correspondence should be addressed to Dr. Ronald K. H. Liem, Department of Pathology, Columbia University College of Physicians and Surgeons, 630 West ture presumably accompanying neuronal maturation. In the pe- 168th Street, New York, NY 10032. ripheral nervous system, this scenariois further complicated by Copyright 0 1990 Society for Neuroscience 0270-6474/90/082735-14$03.00/O the presenceof peripherin, which appearsaround the sametime 2736 Kaplan et al. * dnternexin, a Neuronal IF in the Developing CNS Figure 1. oc-Internexinis an interme- diatefilament protein. a-Intemexin was purifiedfrom a T&on-insoluble,IF-en- riched extract (lane a) by successive hydroxylapatiteand DEAE chromatog- raphyin 8 M urea.The purified material (laneb) readilyformed 10 nm filaments :t uponremoval of the urea, as shown by electronmicroscopy, mag. 113,400x (lanec). cu-Intemexin(I) appearsin cy- Gbf toskeletalextracts as a majorband mi- gratingbelow NF-L, with an apparent molecularweight of 66 kDa; other ma- jor bandsin the extractinclude the pro- teinsofthe neurofilamenttriplet, NF-H (H), NF-M (M), and NF-L (L), aswell asglial filamentprotein, GFA (G). a as NF-M in development (Troy et al., 1990). Unlike the neu- A) containing 0.85 M sucrose and 1 .O% Triton X-100. Unless otherwise rofilament triplet proteins, however, peripherin is up-regulated noted, inhibitors and reagents were obtained from the Sigma Chemical Co. (St. Louis, MO). Homogenates were incubated for 1 hr on ice in following axotomy (Oblinger et al., 1989), suggestingsome role the presence of 0.5 &/ml DNAse I (Boehringer Mannheim, Indianap- for this protein in axon outgrowth. olis. IM and centrifuaed for 5 min at 10.000mm to uelletthe insoluble Apparently, the concept of a “neurofilament triplet” in the filaments. Pellets were washed twice by resuspension in w 10 vol of central nervous system (CNS) may also stand in need of some solution A followed by centrifugation as before. Final pellets were ho- mogenized in approximately equal volumes of either deionized 8 M urea revision. cr-Internexin is a 66 kDa protein that copurifies with or sample buffer (0.0625 mM Tris buffer, pH 6.5, 10% (voVvo1) glycerol, IFS from rat brain, spinal cord, and optic nerve (Pachter and 1% (vol/vol) ,&mercaptoethanol, 1% (vol/vol) sodium dodecyl sulfate, Liem, 1985). Data presentedhere, including polymerization in 0.1% bromophenol blue), and stored at -20°C. vitro, distribution in vivo, and axonal transport, demonstrate Purification and reassembly of Lu-internexin. cu-Intemexin was purified that a-internexin is a neuronal IF protein presentin a wide range from cytoskeletal extracts of rat spinal cord by successive hydroxylapa- tite and DEAE-cellulose chromatography in the presence of 8 M urea, ofmammalian species.These and other data, including sequence as described previously (Pachter and Liem, 1985). For reassembly of analysisof a cDNA clone, conducted in our laboratory (F’liegner purified o-intemexin, urea was removed by dialysis and insoluble ma- et al., 1990)confirm the identity of a-internexin and the “novel terial was pelleted by centrifugation at 100,000 x g for 60 min. Pellets 66 kD NF protein” describedby Chiu et al. (1989). While the were fixed in 1% glutaraldehyde, embedded in plastic resin, sectioned on a Sorvall MT5000 ultramicrotome, and stained with uranyl acetate distribution of ol-internexin in the adult closely parallelsthat of and Reynolds lead citrate for conventional transmission electron mi- the neurofilament triplet proteins, its distribution in the embryo croscopy (Reynolds, 1963). Sections were viewed on a JEOL 1OOC is far more extensive, approaching adult patterns and levels of electron microscope operating at 80 kV. expressionat an earlier age, around embryonic day 16. Im- Antibodies. Two monoclonal antibodies recognizing cu-intemexin, munological and biochemical data presentedhere suggestthat mAb 135 and mAb233, were obtained, according to the method of Koh- ler and Milstein (1976), from 2 separate fusions in the presence of embryonic neuronal IFS contain a higher proportion of cu-in- polyethylene glycol (Gibco Laboratories, Grand Island, NY) of NS-1 ternexin than their adult counterparts, and that the expression mouse myeloma cells with spleen cells from Balb/c mice previously of cy-internexinprecedes that of NF-L in most of the developing immunized with purified ol-internexin.