Microtubule Destabilization and Neurofilament Phosphorylation

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5016-5020, June 1991 Neurobiology Microtubule destabilization and neurofilament phosphorylation precede dendritic sprouting after close axotomy of lamprey central neurons (dendritic identity/neuronal polarity/axonal regeneration/Petromyzon mannus) GARTH F. HALL*t, VIRGINIA M.-Y. LEEO, AND KENNETH S. KosIK* *Department of Neurology (Neuroscience), Harvard Medical School and Center for Neurologic Diseases, Department of Medicine (Division of Neurology), Brigham and Womens Hospital, Boston, MA 02115; and tDivision of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Communicated by Melvin J. Cohen, December 27, 1990 (receivedfor review June 5, 1990) ABSTRACT Axotomy of giant lamprey (Petromyzon man- We have chosen a set ofidentified central neurons [anterior nus) central neurons (anterior bulbar cells) close to their bulbar cells (ABCs)] in the lamprey (Petromyzon marinus), a somata results in ectopic axon-like sprouting from the dendritic primitive vertebrate, to study the events leading up to axon- tips. Such sprouts first appear as swellings at the tips ofa small like sprouting from the dendrites at early times after close subset of dendrites 2-3 weeks after "close" axotomy. We axotomy. Such sprouting has already been investigated in report here that immunocychemical examination of these some detail in lamprey ABCs (8, 9, 12-14), where dendritic swellings reveals a structure and composition that differs from sprouts have been observed to emerge from the dendritic tips that of conventional growth cones; incipient sprouts contain by 2-3 weeks after close axotomy and to grow steadily for many highly phosphorylated neurofilaments (NFs), little tubu- many weeks along typically axonal trajectories. Such sprouts lin, and virtually no stable (acetylated) microtubules (MTs). elongate at mean rates of up to 100 um per day and often The dendrites of anterior bulbar cells after close axotomy also reach lengths of several millimeters. Dendritic sprouting is show pronounced changes in NF protein and tubulin staining accompanied by major cytoskeletal rearrangements in the patterns prior to the emergence of sprouts from the dendrites. dendrites ofthe affected cell by 8-10 weeks after the onset of The amount of tyrosinated tubulin increases greatly; this rise sprouting (14). The cytoskeleton of the sprouts consists is tightly coupled to the appearance of highly phosphorylated almost entirely of neurofilaments (NFs), with very few mi- NFs and the loss of nonphosphorylated NFs in the dendrites. crotubules (MTs). This composition is similar to that ofintact Acetylated tubulin is generally reduced after close axotomy and and regenerating ABC axons, but it is unlike that of either is selectively lost from dendrites that gave rise to sprouts. These intact ABC dendrites (14) or developing axons and their changes indicate that an invasion of the dendrites by phospho- growth cones in other systems, which have few if any NFs rylated NFs may be linked to the destabilization of dendritic (15, 16). This raises the question ofwhether these sprouts had MTs, and in some dendrites this may lead to a marked loss of ceased growing by the time they were examined or if they stable MTs, which is correlated with the emergence ofNF-fled elongate in a manner unlike axons in developing systems or sprouts from the dendritic tips. in vitro. The goals of the present study have been (i) to identify those consequences of close axotomy that may be important in the loss of normal polarity by studying the Axotomy leads to a variety ofchanges in the affected neuron cytoskeletal changes that occur in ABCs before and during [i.e., chromatolysis, an increase in phosphorylated neurofil- the onset of axon-like sprouting from the dendrites and (it) to aments, changes in the electrical properties of the plasma characterize the cytoskeletons of growing sprouts as they membrane, dendritic retraction, etc. (1-8)], some of which emerge from ABC dendritic tips. are probably important preliminaries to axonal regeneration. Most cellular consequences of axotomy are more marked after axotomy close to the soma than after more distant MATERIALS AND METHODS lesions (1). "Close" axotomy has also been shown in several Operations. Close axotomy of ABCs was performed as systems to cause a fundamental cellular change that does not described (8) by hemisection of the hindbrain at a point less occur at all after distant axotomy-i.e., a loss of the ability than 500 um caudad of the rostralmost ABC. This left to regenerate the axon specifically from the axon stump, contralateral ABCs intact to serve as internal controls for resulting in axonal regeneration from the dendritic tips as well effects of surgery (such as deafferentation, local trauma, and as (or instead of) from the axon stump in a number of systems ischemia) and for any section-to-section variations in immu- (6, 9-11). This loss of normal cellular polarity allows one to nocytochemical processing. ask what the mechanisms are that control the site of axonal Immunocytochemistry. Immunocytochemical analysis was regeneration within the cell and also permits one to study the performed on 6-,um transverse sections ofparaffin-embedded earliest events of axonal regeneration in a different context. lamprey brain and spinal cord. Brains were fixed by immer- Because axonal regeneration by definition occurs from an sion of the entire head of anesthetized lampreys in Bouins' injured process (the axon stump), distinguishing early regen- fixative for 6-12 hr at 4°C. These were then washed in 100 erative events from degeneration induced by local injury is mM Tris buffer, dehydrated, impregnated with paraffin, and normally impossible. This difficulty can be circumvented by serially sectioned through the hindbrain. Sections were de- studying ectopic axonal regeneration from uninjured den- paraffinized in xylene and then examined to determine ifthey drites, which permits one to more directly compare the early contained sections through ABC somata and dendrites. They events of axonal regeneration with axonal development. Abbreviations: ABC, anterior bulbar cell; NF, neurofilament; MT, The publication costs of this article were defrayed in part by page charge microtubule; Ac, acetylated; Tyr, tyrosinated; mAb, monoclonal payment. This article must therefore be hereby marked "advertisement" antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5016 Downloaded by guest on September 25, 2021 Neurobiology: Hall et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5017 were then rehydrated, and their endogenous peroxidase activity was quenched with 6% H202 in 80% methanol for 30 min before application of primary antibody (hybridoma supernatant without dilution unless otherwise noted) overnight at 40C. Antibody staining was revealed with an ExtrAvidin- horseradish peroxidase kit (Sigma) by using secondary antibody concentrations, incubation times, and blocking and washing steps as recommended by the manufacturer. Diami- nobenzidine (0.5 mg/ml) was used as the chromagen. Antibodies. The anti-NF monoclonal antibodies (mAbs) employed in this study (RM034, RMdO15, and RM0308) have been extensively characterized (17-20); and the speci- of RM034 to ficity highly phosphorylated NFs, of RMdO15 FIG. 1. Schematic diagram of ABC soma and dendrites (viewed to nonphosphorylated NFs, and of RM0308 to phosphory- as a transverse section at the level ofthe VIIth nerve) illustrating the lation-independent epitopes on NFs in the lamprey has been ABC dendritic field and the criteria used to identify dendritic sprouts established (20). Three mAbs against a-tubulin were used to after close axotomy of ABCs by a hemisection of the brain parallel stain for total a-tubulin (mAb DM1A; ICN) and to differen- to the plane ofview. Higher order ABC dendritic branches have been tiate between relatively stably polymerized MTs and labile omitted for clarity. Six-micrometer sections through the ABC den- MTs or free tubulin. A mAb against acetylated a-tubulin dritic tree (as shown in Figs. 2 and 3) generally did not include (mAb 6-11b-1; kindly provided by G. Piperno of Rockefeller dendrites along their entire length, owing to oblique dendritic tra- University, New York) was used at a dilution of 1:5 (hybrid- jectories, although entire dendrites could usually be assembled in two oma supernatant) to label MTs that are stably polymerized to three adjacent sections. The stippled area (sprout zone) denotes (21, 22). A mAb against tyrosinated a-tubulin (mAb TUB- the region in which swollen profiles were defined as dendritic 1A2; Sigma) was used (ascites, diluted 1:80) to identify sprouts. It was assumed that this sampling region [including most tubulin ABC dendritic tips, but distant from the trajectories of other giant that is not stably polymerized (23, 24). The ability of axons (black circles near the ABC soma)] would prevent the mis- these mAbs to recognize lamprey tubulin specifically was identification of sprouts regenerating from the cut axons of other confirmed on Western blots of lamprey brain cytosolic prep- giant neurons as ABC dendritic sprouts. VIII, VIIth nerve; M, arations run next to extracts of embryonic rat brain (data not midline. Dorsad is toward the top of the figure. shown). Identification of ABC Somata and Dendrites. Normal ABC somata, dendrites, and axons in sections oflamprey brain and morphology

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