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Calpain Activity Promotes the Sealing of Severed Giant Axons (Axotomy͞crayfish͞medial Giant Axon͞squid Giant Axons͞plasmalemmal Repair)

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Calpain Activity Promotes the Sealing of Severed Giant Axons (Axotomy͞crayfish͞medial Giant Axon͞squid Giant Axons͞plasmalemmal Repair) Proc. Natl. Acad. Sci. USA Vol. 94, pp. 4751–4756, April 1997 Neurobiology Calpain activity promotes the sealing of severed giant axons (axotomyycrayfishymedial giant axonysquid giant axonsyplasmalemmal repair) CHRISTOPHER M. GODELL*†,MARK E. SMYERS†,CHRISTOPHER S. EDDLEMAN*, MARTIS L. BALLINGER†, HARVEY M. FISHMAN*, AND GEORGE D. BITTNER*†‡ *Department of Physiology and Biophysics, University of Texas, Medical Branch, 301 University Boulevard, Galveston, TX 77555-0641; and †Department of Zoology, College of Pharmacy and Institute for Neuroscience, University of Texas, Austin, TX 78712 Communicated by Donald Kennedy, Stanford University, Stanford, CA, February 28, 1997 (received for review May 9, 1996) ABSTRACT A barrier (seal) must form at the cut ends of (2) and MGAs (1) transected in Ca21-free salines do not seal.] a severed axon if a neuron is to survive and eventually According to our electrophysiological measures (decay of regenerate. Following severance of crayfish medial giant injury current, Ii, and resting membrane potential, Vm) and axons in physiological saline, vesicles accumulate at the cut optical measures (dye exclusion), we find that exogenous end and form a barrier (seal) to ion and dye diffusion. In calpain, but not chymotrypsin, induces sealing of transected contrast, squid giant axons do not seal, even though injury- squid GAs when added to physiological salines. Calpain does induced vesicles form after axonal transection and accumulate not appear to enhance vesicle formation in squid GAs, but at cut axonal ends. Neither axon seals in Ca21-free salines. rather enables existing vesicles to form a seal. According to The addition of calpain to the bath saline induces the sealing these same measures, crayfish MGAs normally seal in physi- of squid giant axons, whereas the addition of inhibitors of ological salines, but crayfish MGAs do not seal when calpain activity inhibits the sealing of crayfish medial giant transected in physiological salines containing exogenous in- axons. These complementary effects involving calpain in two hibitors of calpain activity (leupeptin, human calpastatin pep- different axons suggest that endogenous calpain activity pro- tide, calpeptin, endogenous rabbit calpastatin, iodoacetamide, motes plasmalemmal repair by vesicles or other membranes and monoclonal antibodies to calpain). However, crayfish which form a plug or a continuous membrane barrier to seal MGAs will seal in Ca21-free solutions containing exogenous cut axonal ends. proteases (bromelian, papain, dispase, trypsin, chymotrypsin), which do not require Ca21 for activation and which (unlike The rapid (within 60 min) repair of unmyelinated crayfish calpain) are not normally found in axons. These and other data medial giant axons (MGAs) (1) and myelinated earthworm (11) suggest that endogenous calpain enhances the sealing of MGAs (2) is associated with the accumulation of vesicles and severed invertebrate and vertebrate (including mammalian) other membranous material at the cut axonal ends. In contrast, axons primarily by inducing junctional complexes in an accu- squid giant axons (GAs) do not seal, even though injury- mulation of vesicles or by enhancing the formation of a induced vesicles form after transection and accumulate at cut continuous membrane, rather than by enhancing vesicle for- axonal ends (3). Calpain (4) is a calcium-activated neutral mation, vesicle movement, or axonal closure by degrading protease endogenous to squid GAs (5), other axons (4), and cytoskeletal proteins. other cell types (4, 6) that could enhance or inhibit one or more stages (vesicle formation, movement, aggregation, membrane MATERIALS AND METHODS fusion, etc.) of the sealing process. Calpain and other proteases degrade cytoskeletal proteins such as neurofilaments (4, 6, 7, As previously described (2), squid GAs dissected from Loligo 8), which help maintain axonal diameter and shape (9). Such pealei or Sepioteuthis lessoniana were placed in artificial sea degradation might produce a complete or partial collapse of water (physiological saline for squid): 430 mM NaCl, 10 mM the cut end (10, 11), thereby inducing sealing by enhancing the KCl, 10 mM CaCl2, 50 mM MgCl2, and 5 mM TriszCl (pH 7.4), interactions between vesicles that form a seal by themselves 974 mOsm (osmolality). Divalent-cation-free salines consisted andyor that form a continuous membrane at the cut end. (In of 426 mM NaCl, 10 mM KCl, 110 mM tetramethylammonium this paper, the term ‘‘complete constriction or complete chloride, 5 mM TriszCl, 1 mM EDTA, and 1 mM EGTA (pH collapse’’ is used to describe 100% closure of a cut axonal end; 8), 972 mOsm. MGAs were dissected from crayfish (Procam- the term ‘‘partial constriction or partial collapse’’ is used to baris clarkii) and placed in various salines, as described in the describe any constriction that leaves an opening at the cut end.) preceding paper (1). Alternatively, calpain or other proteases might inhibit sealing Use of a vibrating probe, intracellular electrodes, differen- by repressing the formation of vesicles or a continuous mem- tial interference contrast (DIC) optics, and fluorescence and brane or by degrading cytoskeletal proteins (microtubules, confocal fluorescence imaging were as described (1). In this actin, molecular motors, etc.), which might affect the move- paper, relative measures of injury current density (Ii) and ment of injury-induced or other vesicles to the cut axonal end. control current density (Ic), measured for the intact axon prior We examined the effects of calpain and other proteases on to its transection, are denoted by the primed symbols I9i and I9c; the sealing of two giant axons: the crayfish MGA, which I9i and I9c are normalized with respect to the first value of Ii normally seals after severance in physiological saline (see acquired 3–5 min after transection. According to this defini- preceding paper, ref. 1), and the squid GA, which normally tion, the initial value of I9i always equals 100%. An axon was does not seal after severance in physiological saline (2). [GAs considered to seal electrically when Ii decayed to the upper end 2 of the range of values recorded for Ic (25 mAycm for GAs and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: DIC, differential interference contrast; GA, giant accordance with 18 U.S.C. §1734 solely to indicate this fact. axon; MGA, medial giant axon; Vm, membrane potential; Ii, injury current density; Ic, control current density; FITC, fluorescein isothio- Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA cyanate. 0027-8424y97y944751-6$2.00y0 ‡To whom reprint requests should be addressed at: Department of PNAS is available online at http:yywww.pnas.org. Zoology, University of Texas, Austin, TX 78712-1064 4751 Downloaded by guest on September 30, 2021 4752 Neurobiology: Godell et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2 15 mAycm for MGAs). The largest Ic values were more than after transection (2). Vm then began to decline steadily for 20 times smaller than the smallest Ii values recorded after 60–120 min after transection (Fig. 1, Table 1), almost certainly transection. due to degenerative changes in the GA (2, 12). As previously Dyesandothersubstanceswereobtainedasfollows:a-chymo- reported (2), Ii and Vm at 90–150 min after transection were trypsin, bromelian, calpain, cytochalasin E, iodoacetamide, significantly different (P , 0.001) from control values (Table leupeptin, papain, protease inhibitor (endogenous rabbit cal- 1). According to these combined measures, GAs transected in pastatin), tetramethylammonium chloride, and trypsin (Sig- physiological saline did not seal within 90–120 min (Table 2). ma); calpeptin and taxol (LC Laboratories, Woburn, MA); DIC (Fig. 2A) or confocal fluorescence (Fig. 2B) images of fluorescein isothiocyanate (FITC)–dextran, Lucifer Yellow, GAs in vitro and photomicrographs of thick sections (Fig. 2B Oregon Green-dextran, pyrene, Texas Red–dextran, tetram- Inset) or electron micrographs of thin sections of fixed GAs ethylrhodamine–dextran (Molecular Probes); calpeptin (Cal- showed that cut ends transected in physiological salines were biochem); human calpastatin peptide (Research Biochemicals, not completely closed and contained loosely packed vesicles Natick, MA); dispase (Boehringer Mannheim); calpain anti- 0.1–50 mm in diameter at 60–120 min post-severance. As body (N. Banik, Medical University of South Carolina, previously reported (2), inspection of electron micrographs Charleston). from over 10 GAs showed that the axolemma and glialemma were disrupted for 50–200 mm from the cut end. When RESULTS AND DISCUSSION transected and maintained in physiological saline for 60–90 min before adding hydrophilic dyes to the bath, GAs did not We measured the Ic and membrane potential (Vm) of control exclude hydrophilic dye (did not seal) at 90–120 min post- (intact) squid GAs in physiological saline (Fig. 1, Table 1). severance (Fig. 2 B and C; Table 2). Orthograde and retro- Intact GAs, 300–600 mm in diameter, were surrounded by a grade axonal transport ceased in GAs (n 5 3) at 60 min nonmyelinated glial sheath 5–10 mm wide consisting of glial posttransection. I9c of intact GAs (n 5 5) measured for up to and collagen layers (Fig. 2B Inset) (12, 13). The axoplasm of 120 min was not significantly (P . 0.05) different in physio- intact GAs contained neurofilaments, microtubules, smooth logical saline compared with that in saline containing calpain endoplasmic reticulum, mitochondria, and some vesicles ,1 (0.02 activity unityml). Intact GAs (n 5 3) excluded hydro- mm in diameter. Ic of intact GAs (n . 10) ranged from 4 to 12 philic fluorescent dye and exhibited orthograde and retrograde 2 mAycm and was directed outwardly (2). Ic did not change for axonal transport for up to 120 min after adding calpain. 120 min unless the GA was accidentally damaged, in which case For 3–20 min posttransection, the rates of decay of I9i for GAs Ic increased 20–200 times as rapidly as the new measurement transected in physiological saline containing calpain were not could be made.
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