Jorrriiul ~ ~ / N i ~ u r o c l r r i ? i I . s / r ~ ~ Raven Press. Ltd., New York 0 1994 International Society for Neurochemistry

Rapid Communication

Calcium-Mediated Degeneration of the Axonal Cytoskeleton in the Ola Mouse

*?Jonathan D. Glass, *Brenda L. Schryer, and *$John W. Griffin

Depurtr?ient.s of *Neiirology. tPatho1og.v (Netiropatholo~v). and $Neuroscience, The Johns Hopkins University School qf Medicine, Bull imore, Murylund. U.S.A .

Abstract: The C57BL/Ola (Ola) mouse is a mutant substrain in which transected axons undergo very slow Wallerian degenera- tion. Because axonal degradation during Wallerian degeneration is calcium dependent, we tested whether Ola axons are suscepti- ble to calcium-mediated axonal degeneration by comparing neuro- filament degradation between Ola and C57BL/6 mice in sciatic nerve explants. Using immunoblot analysis of neurofilament deg- radation and electron microscopy we found that as in normal axons, axonal degeneration in the Ola is calcium dependent. How- ever, when compared with normal animals, higher levels of cal- cium were required for complete degradation of neurofilaments in Ola nerve, suggesting a relative insensitivity to calcium-mediated degeneration in the Ola. We conclude that calcium-activated pro- teases are present and active in Ola axons but that higher levels of calcium are required to accomplish complete axonal degrada- tion. These results suggest a possible mechanism for prolonged survival of transected Ola axons and provide potential insight into the pathophysiology of axonal degeneration in injury and disease. Key Words: Calcium-Calpain-Axon-Ola mouse-Neurofila- ments-Wallerian degeneration. J. Neurochem. 62,2472-2475 (1 994).

Axons contain calcium-activated neutral proteases (cal- pains), which have been implicated as playing a major role in axonal degeneration (Schlaepfer, 1974; Zimmerman and Schlaepfer, 1982; Kamakura et al., 1983). Previous studies by Schlaepfer and colleagues have demonstrated that neuro- filaments (NFs), the major structural components of the axonal cytoskeleton, are readily degraded by calpains in the presence of sufficient levels of axonal calcium, resulting in the loss of immunoreactivity for NFs (Schlaepfer and Micko, 1979; Zimmerman and Schlaepfer, 1982; Schlaepfer et al.. 1984). The structural correlate of this bio- chemical change is the replacement of axoplasm by amor- phous granular debris (Schlaepfer, 197 1, 1974, 1977: Schlaepfer and Micko, 1978; Schlaepfer and Hasler, 1979). This morphological and immunochemical pattern is also seen within the early phases of Wallerian degeneration (Schlaepfer and Micko, 1978: Schlaepfer et al., 1984, 1985). the axonal degeneration triggered by section of nerve fibers.

The C57BL/Ola (Ola) mouse is a mutant substrain in which transected nerves in the PNS and CNS undergo very slow Wallerian degeneration (Lunn et al., 1989; Glass and Griffin, 1991: Ludwin and Bisby, 1992). The only pheno- typic difference between Ola and standard C57BL/6 mice is

the rate of Wallerian degeneration, and it has been demon- strated that the phenotype is not related to systemic abnor- malities in the Ola mouse but is an intrinsic characteristic of the axon (Perry et al., 1990; Glass et al., 1993). The slow Wallerian degeneration trait has been mapped to chromo- some 4 (Lyon et al., 1993). We have shown that transected axons from Ola mice not only survive for prolonged periods in vivo but also remain intact longer than axons from C57BL/6 mice when transected as cultured neurites (Glass et al., 1993) or in organ cultures of adult sciatic nerves (J. D. Glass et al., unpublished data).

An understanding of why Ola axons survive longer than normal axons will certainly provide insight into the normal mechanisms of axonal degeneration. Because axonal degra- dation during Wallerian degeneration is a calcium-me- diated event, prolonged survival of transected axons in the Ola could involve alterations in calcium entry. buffering, or calcium-sensitive effector mechanisms. Because calpain is the effector molecule most likely to be involved in axonal degeneration, we tested the physiologic activity of axonal calpain in the Ola mouse by comparing axonal degenera- tion in response to calcium in C57BL/6 versus Ola mice in short-term nerve cultures.

MATERIALS AND METHODS Ola mice were obtained from a breeding colony generated

from original breeding pairs purchased in 1989 from Har- lan/Olac (England) and maintained at The Johns Hopkins University School of Medicine. Standard C57BL/6 mice used for comparison were purchased from Harlan (Freder- ick, MD, U.S.A.). All procedures and animal housing and care were according to AAALAC-approved protocols. The

Resubmitted manuscript received February 14, 1994; accepted February 16, 1994.

Address correspondence and reprint requests to Dr. J. D. Glass at Department of Neurology, The Johns Hopkins University School of Medicine, Pathology Building 627.600 North Wolfe Street, Bal- timore, MD 21287-6965, U.S.A.

Abbreviations used. NF, neurofilament; NF-H, NF-L, and NF- M. 200-, 68-, and 160-kDa neurofilament, respectively; Ola, C57BL/Ola.

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AXONAL DEGENERATION IN THE OLA MOUSE 24 73

animals were killed by cardiac transection while under me- thoxyflurane inhalation anesthesia. Short-term organ cultures

Ten-millimeter segments of sciatic nerve were removed using sterile technique and were quickly placed in 7-ml plas- tic scintillation vials containing the defined media ( 1 30 mM NaCI, 4 mMKC1, 1 mMMgCI,, 14 mMglucose, and 5 mM Tris-HCI buffer, pH 7.4) with or without CaCI,, EGTA, or Triton X-100. Addition ofsodium cyanide in the absence of axolemmal disruption was used to test whether inhibition of oxidative metabolism could initiate the degradative re- sponse. Defined calcium concentrations were measured us- ing a calcium-sensitive electrode. Short-term cultures were done either at room temperature or in a 37°C water bath for 2 h. Experiments requiring “freeze-thaw’’ for disruption of the axolemma were performed by placing the vial in pow- dered dry ice for 10 min, allowing it to thaw on the bench- top, and then repeating. After 2 h in culture the nerve seg- ments were fixed for morphology or frozen at -70°C for western blot or in some experiments were cut in half so that both morphology and immunoblot could be compared in the same segment.

Morphological analysis was camed out by fixing in 5% glutaraldehyde/O. 1 M phosphate buffer, postfixing in os- mium tetroxide, and embedding in Embed 8 12/Araldite 502. Thin sections for electron microscopy were stained with lead citrate and uranyl acetate and examined on a Hi- tachi model H-600 electron microscope. Immunoblot analysis

As demonstrated previously (Schlaepfer and Micko, 1978, 1979), we found that an excellent correlate of granu- lar disintegration ofthe cytoskeleton, the morphologic deter- minant of axonal degeneration, is the loss of NF immunore- activity on western blots. Nerve segments were homoge- nized in glass-on-glass homogenizers containing 60 j ~ l of urea gel sample buffer (6 M urea, 2% sodium dodecyl sul- fate, 10% glycerol, 0.1 % bromphenol blue, and 10% p-mer- captoethanol in 0.5 M Tris buffer, pH 6.8). Samples were boiled and centrifuged, and a 5-111 aliquot was loaded onto a single lane of a vertical polyacrylamide minigel and electro- phoresed at constant voltage. Separated proteins were trans- ferred to nitrocellulose over - 1 h at constant current. De- tection of NF proteins was by standard immunochemical methods using monoclonal antibodies that are relatively spe- cific for the different NF epitopes: SMI 31 (Sternberger Monoclonals) for phosphorylated 200-kDa NF epitopes (NF-H) (l:lO,OOO), and NF 160 (NF-M) and NF 68 (NF-L) (Boehringer Mannheim) for the 160-kDa (1 : 1000) and 68- kDa ( 1 : 1000) epitopes, respectively. Either a single antibody or a cocktail of all three antibodies was used. Secondary antibodies were conjugated directly to horseradish peroxi- dase, and color detection used, 3,Y-diaminobenzidine as the substrate.

Immunoreactive bands for 68- and 160-kDa NF were quantified by laser densitometry (Molecular Dynamics; OD range, 0-4.095). A comparison of the relative band intensi- ties of C57 versus Ola nerves was generated by calculating a densitometric ratio, C57/01a, for each calcium concentra- tion.

RESULTS Calcium-mediated degeneration in short-term organ cultures

Sciatic nerve segments from age-matched animals were placed in media either at room temperature or at 37°C for

- - 68 . _ c 57

4- -0 68 - -- OLA 4 7 .5 .4 .3 .2 . I 0

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FIG. 1. lmmunoblots stained with either a cocktail of antibodies against NF epitopes (a) or single antibodies against NF 68 (b) or NF 160 (c) Each lane represents a single nerve segment incu- bated in media containing 1% Triton and calcium at the concen- tration designated along the bottom (millimolar) The blots shown in (a) demonstrate that the 200-kDa band (stained with SMI 31) is relatively resistant to degradation even at the highest calcium concentrations, although it does degrade into two bands at >O 2 mM. At 0 2 mM the 68-kDa band is gone in C57BL/6 but is par- tially retained in Ola (arrowhead) With degradation, new low-mo- lecular-mass bands (45-60 kDa) are formed in both strains, these are likely NF breakdown products NF degradation is directly compared in (b) and (c) at multiple [Ca”] between Ola (right lane of each pair, “0” and C57BL/6 (left lane of each pair, “c”) The difference in calcium threshold for NF degradation is demon- strated for NF-L (b) and NF-M (c) Faint bands in Ola are seen out to 0 30 mM for both epitopes Low-molecular-mass NF bands representing breakdown products are seen with the NF 68 anti- body and first appear at 0 10 mM (b)

exactly 2 h. To maintain the validity of comparisons be- tween substrains, the nerve segments from Ola and C57BL/ 6 mice were assayed in parallel using the same batches of freshly prepared media. Entry of extracellular calcium into the axon was achieved by the freezerthaw protocol or by addition of 1% Triton X-100 to the media (Schlaepfer and Micko, 1979). Initial experiments showed that axoplasmic proteolysis, reflecting axolemmal disruption and calcium entry, was equally successful with either technique, and so 1% Triton was used in subsequent experiments. Experi- ments were done in quadruplicate.

Western blots The nerve segments were placed in media with no added

calcium (0 mM) or media containing 0.10,O. 15,0.20,0.25, 0.30, 0.40, or 0.50 mM calcium chloride. In both strains there was no degradation over 2 h of NF proteins without the freeze/thaw protocol or addition of 1% Triton. With axolemmal disruption in both strains, the immunoreactive bands for the 68- and 160-kDa proteins consistently disap- peared at calcium concentrations of 20.30 &(Fig. 1). The degradation of these proteins was inhibited by addition of the calcium chelator EGTA, and the threshold for degenera- tion was not altered by incubation at 37°C (data not shown). As previously shown (Schlaepfer and Micko, 1978; Schlaepfer et a]., 1984), the 200-kDa band stained with the SMI 3 1 antibody was relatively resistant to complete degra-

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DISCUSSION The present data demonstrate that calpains are present

and active in Ola axons. These enzymes accomplished degra- dation of NFs by morphological and immunochemical crite- ria in both substrains in a non-temperature-dependent fash- ion. Granular disintegration of the cytoskeleton by electron microscopy was verified on western blots by the loss of im- munoreactive bands for the NF-M and NF-L epitopes. The 200-kDa band, as revealed by an antibody recognizing highly phosphorylated forms of NF-H, persisted even when NFs were absent on electron microscopy. This phenome- non was previously recognized (Schlaepfer and Micko, 1978, 1979) and is likely related to an interaction of the NF-H subunit with calmodulin, which inhibits calpain-me- diated degradation of NF-H (Johnson et al.. 1991). Because of the persistence of NF-H immunoreactivity, comparisons of NF degradation were restricted to the NF-M and NF-L bands.

Previous studies demonstrating that calcium is necessary for granular disruption of axons (Schlaepfer, 197 I , 1974, 1977; Schlaepfer and Hasler, 1979: Schlaepfer and Micko, 1979; Waxman et al., 1993) did not identify a concentration threshold for activation of axonal calpains. We found that in nerve fibers from standard C57BL/6 mice the calcium concentration necessary for complete loss of NF-M and NF- L bands was 0.20 mM. This threshold was consistent be- tween experiments and was not affected by temperature. In Ola nerves. however, the calcium concentration required to achieve complete degradation of NF-M and NF-L proteins was consistently higher. Although there was partial degrada- tion of NF proteins at 0.20 mM as suggested by the relative loss of intensity of immunoreactive bands and the appear- ance of new low-molecular-mass bands (Schlaepfer et al., 1985), a small amount of immunoreactive NF-M and NF-L proteins remained even at 0.30 mM calcium. Direct com- parisons of immunoreactive bands from C57BL/6 and Ola nerves subjected to the same experimental conditions and electrophoresed on adjacent gel lanes demonstrated a statis- tically significant difference at 0.20 mM.

i (C57 IOla)

mean +/- SD o.6

0.4

0.2 1

I. 0

I. t: I.

0.0 0 .10 .15 .20

CALCIUM CONCENTRATION (mM)

FIG. 2. Comparisons of C57 versus Ola band intensities for NF-L and NF-M. The difference between C57BL/6 and Ola breakdown is demonstrated at 0.20 mM (p < 0,001).

dation even at the highest calcium levels tested (Fig. la). Appearing on the blots from partially or fully degenerated nerves were new low-molecular-mass bands (-45-60 kDa) that stained with the NF-L antibody (Fig. la and b). These bands are likely to represent NF cleavage products (Schlaepfer et al., 1984, 1985). They were most prominent in the nerves maintained in 0.20-0.40 mM calcium and largely disappeared at 0.50 mM calcium. Addition of so- dium cyanide in the absence of at least 0.20 mM calcium did not cause degradation of NF proteins (data not shown).

Even though the nerves from both substrains showed de- generation in the presence of high calcium concentrations, there was a difference between C57BL/6 and Ola in the calcium concentration necessary for complete NF degrada- tion. Nerve segments from C57BL/6 mice showed loss of NF epitopes at 20.20 mM, whereas Ola nerve segments retained NF immunoreactivity to the 68- and 160-kDa epi- topes at calcium concentrations of 0.20 and 0.25 mM (Fig. 1 b and c). The intensity of these immunoreactive bands was reduced compared with the bands at t0.20 mM calcium, suggesting partial destruction of these epitopes. These find- ings were consistent between experiments. The densitomet- ric ratios comparing band intensities of C57 to Ola (Fig. 2 ) showed a significant decrease in this ratio at 0.20 mM when compared with 0, 0.10, and 0.15 mM (p < 0.001 by ANOVA).

Electron microscopy The specimens of nerve examined under electron micros-

copy corroborated the findings on western blots. Nerve seg- ments treated with freeze/thaw or with 1% Triton showed disruption of myelin sheaths and partial or complete de- struction of the axolemma. NFs were preserved when seg- ments did not undergo freeze/thaw or exposure to Triton even in media with >0.20 mMcalcium. NFs were also pre- served with addition of 1% Triton or freeze/thaw when the calcium concentration in the media was low, i.e., <200 p M or with addition of EGTA. There was granular disintegra- tion of the cytoskeleton with destruction of NFs (as well as other cytoskeletal constituents) with the combination of ax- olemmal disruption and high calcium concentration, i.e., 20.20 mM. As suggested by the immunoblot results, there was relative preservation of NFs in Ola versus C57 nerves at 0.20 mM (Fig. 3).

FIG. 3. Representative axonal profiles from (A) Ola and (8) C570L/6 nerves incubated in Triton and 0.20 mM calcium. The Ola axon shows intact neurofilaments, whereas the C57BL/6 axon is reduced to amorphous granular debris. Note the deter- gent-disrupted myelin in each. A: X21,OOO. B: X16,500.

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AXONAL DEGENERATION IN THE OLA MOUSE 24 75

The inability of sodium cyanide to initiate the loss of NFs in the absence of sufficient calcium suggests that energy fail- ure alone does not account for cytoskeletal disruption. This has also been demonstrated in a system where anoxic injury to cultured optic nerves fails to cause axonal degeneration in the absence of extracellular calcium (Waxman et al., 1993).

The difference in the threshold calcium concentration needed for complete degeneration of NFs in Ola and C57BL/6 axons could be due to differences at various func- tional levels. An obvious possibility is that axonal calpains in Ola mice are less sensitive than in normal mice to intra- cellular levels of calcium. Alternatively, the calcium homeo- static mechanisms that prevent activation of calpains under normal conditions, such as the interaction with the endoge- nous inhibitor calpastatin (Murachi, 1989), may be “over- active” in the Ola. Elevated amounts of calpastatin relative to the amount of calpain might create a milieu requiring higher than normal levels of calcium for activation of cal- pain. Alteration in the relative amounts of neuronal calpain and calpastatin has been suggested as the mechanism of neuronal degeneration seen in the mutant Mnd mouse (Li et al., 1993).

Because of its unique characteristic of “slow” Wallerian degeneration in a higher-order mammal, the Ola mouse provides an experimental system to investigate the mecha- nisms underlying normal axonal degeneration in mammals. A clear understanding of the biological differences between OIa and normal mice should direct us toward the mecha- nisms of axonal degeneration in experimental systems and in disease.

Acknowledgment: We thank David Figueroa and James Watkins for technical assistance, Edwin George and Jeffrey Rothstein for helpful discussion, and Rod Graham for rnan- uscript preparation. This work was supported by grants NS 14784, NS 01577, and POI 22849 from the National Insti- tutes of Health.

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