68

Brain Research, 580 (1992) 68-80 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 17707

Maintenance and synthesis of proteins for an anucleate axon Rebecca A. Sheller a'c and George D. Bittner a'b'c Department of ~Zoology, bCollege of Pharmacy and Clnstitute for Neuroscience, The University of Texas at Austin, Austin, TX 78712 (USA) (Accepted 24 December 1991) Key words: Anucleate; Axotomy; Crayfish; Glia; Local protein synthesis; Protein transfer

The anucleate (distal) segment of a crayfish medial giant axon (MGA) remains intact for months in vivo after severing the axon from its cell body, a phenomenon referred to as long-term survival (LTS). We collected axoplasm from chronic anucleate MGAs by perfusing 2-cm lengths of axons with an intracellular saline. This axoperfusate was analyzed by SDS-PAGE and silver stained. Axoperfusate proteins from intact MGAs and from chronic anucleate MGAs exhibiting LTS for up to 6 months were the same. Furthermore, immunoreactive levels of actin and fl-tubulin were similar in axoperfusates from intact and chronic anucleate MGAs. This maintenance of proteins in chronic anucleate MGAs must be due to a lack of protein degradation and/or to local protein synthesis by a source other than the cell body. To investigate local protein synthesis in vitro, we added [aSS]-methionine to the extracellular saline surrounding intact and chronic anucleate MGAs. After 4- to 6-h incubations, radiolabelled proteins were detected in axoperfusates analyzed by SDS-PAGE and fluorography. The similarity between radiolabelled proteins in axoperfusates and MGA glial sheaths indicated a glial origin for the radiolabelled axoperfusate proteins. Various observations and control experiments suggested that glial-axonal protein transfer occurred by a physiological process. Glial-axonal protein transfer may contribute to the maintenance of proteins during LTS of chronic anucleate MGAs. INTRODUCTION In contrast to anucleate (severed distal) segments of mammalian axons which undergo Wallerian degeneration within several days, chronic anucleate axons in many invertebrates and some lower vertebrates exhibit longterm survival (LTS) for weeks to years after severance from their cell bodies 3. In the crayfish CNS, medial giant axons (MGAs) exhibit LTS in vivo for 50-200 days after lesioning 2'4'5'46. LTS of chronic anucleate M G A s is characterized by the presence of intact mitochondria, microtubules, and smooth endoplasmic reticulum 2 and by physiological values for resting potentials, action potentials and conduction velocities 5'46. Chronic anucleate M G A s gradually decrease in diameter and usually completely degenerate 250-300 days after lesioning 2'4'5'46. In order to evaluate LTS biochemically, we obtain axoplasm from intact and chronic anucleate M G A s by perfusing segments of these axons with intracellular saline and collecting the axoperfusates. Axoperfusate proteins are separated by S D S - P A G E and silver-stained. All axoplasmic proteins detected by silver stain, including actin and fl-tubulin, are maintained in chronic anucleate M G A s severed for up to 6 months. In order to maintain axoplasmic proteins, chronic anucleate M G A s must retain existing proteins (e.g. slow protein turnover) and/or obtain newly synthesized proteins (e.g. axoplasmic pro-

rein synthesis or intercellular transfer of proteins from adjacent cells). Glial-axonal protein transfer was demonstrated over a decade ago for intact (not chronic anucleate) squid giant axons 11'2°-22. In these previous studies, the squid giant axons were incubated in radiolabelled amino acids for several hours before axoplasm was collected either by extrusion 2°-22 or perfusion 11. Many radiolabelled proteins were detected in the axoplasm of the squid giant axon. Some of the proteins transferred from the glia to the giant axon have since been reported to be heat shock proteins (HSPs) 38'4°. Therefore, one function for glialaxonal protein transfer in the squid may be to provide a stressed axon with an immediate source for HSPs. In the crayfish, glial-axonal protein transfer has been suggested by autoradiographic techniques for intact and chronic anucleate M G A s 29. Due to the limitations of autoradiography, this previous study on crayfish M G A s 29 was unable to determine the number or molecular weights of transferred proteins. In the present study we use the technique of axonal perfusion to obtain axoplasm and biochemical techniques to investigate local protein synthesis in intact and chronic anucleate MGAs. Since the M G A cell body synthesizes proteins which are transported along the axon 1°'43, we isolate the M G A cell body from a 2-cm axonal segment in order to study local axonai protein synthesis. We incubate this 2-cm M G A

Correspondence: R.A. Sheller, Department of Zoology, The University of Texas, Austin, TX 78712, USA. Fax: (1) (512) 471-9651.

69 segment in [35S]methionine ([aSS]met) in vitro, and obtain radiolabelled proteins by internally perfusing this MGA segment. Radiolabelled proteins in axoperfusates are the same for intact and chronic anucleate MGAs. We also find that radiolabelled proteins in axoperfusates are similar to radiolabelled proteins in glial sheaths. These data are consistent with the hypothesis that many species of proteins synthesized in glia are transferred to adjacent MGAs by a physiological process. Such transfer might contribute to the LTS of chronic anucleate MGAs. MATERIALS AND METHODS

Transection of MGAs Crayfish (Procambarus clarkii) 5-9 cm in body length were purchased from local suppliers and kept in large community tanks at 15-18°C. The water and food supply (cat chow and grated carrots) were changed every other day. A pair of MGA cell bodies (right and left) are located in the most rostral ganglion (supraesophageal) of the ventral nerve cord (VNC) and the MGAs extend the entire length (4-7 cm) of the VNC on its dorsal surface (Fig. 1). Approximately 25 animals were anesthetized by chilling to 4°C before a small opening in the cuticle of each crayfish was created to expose the circumesophageal connectives just caudal to the supraesophageal ganglion. We used a small glass hook to lift the left circumesophageal connective which was then completely severed with iridectomy scissors. This operation produced a long (3-6 cm) anucleate segment of the left MGA while leaving the right MGA intact as a sham-operated control. After severing the left circumesophageal connective, the cuticle was replaced and the lesioned crayfish was returned to a community tank. The survival rate of lesioned animals maintained for up to 6 months was 60-80% of the survival rate of non-lesioned animals.

Perfusion of MGAs Perfusion of an MGA required 2 cannulae (Fig. 2), each made from a 1.2 mm diameter glass capillary and pulled to have a long, tapered tip bent at a 45° angle. One cannula, used for injection, was filled at the tip with 25/~1 MGA intracellular saline modified from Shrager et al. 37 as follows: 109 mM KF, 37 mM K 3 citrate, 15 mM NaC1, 96 mM mannitol, 5 mM HEPES, pH 7.4. The rest of the injection cannula was filled with mineral oil. The other cannula, used for collection, was entirely filled with mineral oil. The two glass cannulae were connected to polyethylene tubing filled with

mineral oil and the tubing was connected to two 50-ml glass sytinges, also filled with mineral oil. Glassware, tubing, and mineral oil were sterilized in an autoclave for 20 min. MGA intracellular saline was sterilized by filtration through a 0.2-/~m cellulose nitrate filter. Prior to cannulation and perfusion, crayfish (n > 70) were anesthetized by chilling to 4°C before removing the entire VNC containing intact and/or chronic anucleate MGAs. The VNC was placed in a Sylgard coated petri dish containing crayfish extracellular saline42 at room temperature (20-22°C) and pinned dorsal side up so that the paired MGAs were easily visible under a dissecting microscope. A small well (2 cm long, 0.7 cm wide, and 0.2 cm deep) was cut into the Sylgard layer of the preparation dish (Fig. 2). The VNC was positioned so that a 2-cm length extending from the circumesophageal region to the abdominal region lay within the well; the VNC rostral to the 2-cm length and the VNC caudal to the 2-cm length lay outside the well. A Vaseline barrier was carefully placed around the well and the 2 cm length of VNC inside the well was covered with 250 ~1 of extracellular well saline (EWS). Each of the rostral and caudal segments of VNC outside the well were also covered with about 250 ~l of extracellular saline. The Vaseline barrier separated the saline inside the well from the saline outside the well (see Results). The collection and injection cannulae were inserted into an MGA 100-200 ~m in diameter. An incision was made in the rostral region of the axon outside the well (Fig. 2) and the collection cannula (50-60 ~m in tip diameter) was inserted through this incision into the axon. A small mineral oil bubble (0.01-0.03 ~l) was injected from the collection cannula into the axon, the cannula tip was advanced approximately 2-3 mm past the bubble and the cannula was tied in place with 9-0 surgical silk. The mineral oil bubble and the silk ligature prevented axoperfusate from leaking out of the MGA during collection. The mineral oil bubble remained in place during the perfusion. A second incision was made in the same MGA in the caudal region outside the well and the injection cannula (20-30 ~m in tip diameter) was inserted through this incision. The injection cannula tip was advanced 2-8 mm past the cannulation incision, a small amount of intracellular saline was injected into the MGA, and the injection cannula was also tied in place with 9-0 surgical silk. The distance between the tips of the two cannulae was slightly greater than 2 cm. Perfusion was initiated with positive pressure at the injection cannula and negative pressure at the collection cannula. When viewed through a dissection microscope, we observed axoperfusate to move rapidly within the axon from the caudal injection cannula into the rostral collection cannula. Axoperfusate in the collection cannula formed an obvious boundary with the displaced mineral oil. The typical volume of axoperfusate collected from a 2-cm length of MGA was 25/~1 (the original volume of axoplasm from a 2-cm length of MGA was estimated to be 0.15 pl). The amount of axoperfusate collected never exceeded the amount of intracellular saline injected.

RadiolabeUing of MGAs Only the 2 cm VNC segment inside the Sylgard well was incubated with [35S]met. The incubation began when the original EWS was exchanged for 250 ~1 of EWS containing [aSS]met (0.6-0.7 ~Ci/~l). Axoperfusate was collected from the 2-cm MGA segment inside the well by one of two paradigms described below.

(1) Delayed-collection paradigm: add radiolabel to incubation well, wait 4-6 h and cannulate MGA for perfusion. In the majority

Fig. 1. Histological cross section of a pair of intact MGAs at the dorsal surface of the abdominal VNC. Bar = 24 #m.

of radiolabelling experiments (n = 31), the 2-cm length of VNC inside the well was incubated in EWS containing [35S]met for 4-6 h before the MGA was ever cannulated. At the end of the incubation period, a 100-~1 aliquot was collected from the radiolabelled EWS. The VNC segments inside and outside the well were then rinsed at least 5 x with 250 ~l aliquots of unlabelled extracellular saline. The collection and injection cannulae were inserted into the MGA as described above. The MGA was usually perfused for

70 10-15 min with about 25/~1 of intracellular saline. (Sometimes, the MGA was perfused with only 1-8 kd of intracellular saline.) To inhibit translation in some preparations, we added 10-4 anisomycin to the EWS 30 min prior to and during the incubation with [aSS]met. The cannulae tips were usually inserted into the MGA outside the incubation well as described above and the distance between the cannulae tips ranged from 2 to 3 cm with an average of 2.4 cm. Sometimes, one or both cannula tips were inserted into the M G A inside the incubation well after the radiolabelled EWS had been replaced with unlabelled EWS; the distance between the tips of these cannulae ranged from 0.4 to 2.3 with an average of 1.4 cm.

adigm was used as a control to test for axoplasmic protein synthesis and artifactual leakage of radiolabelled proteins. Axoplasmic protein synthesis was less likely to occur in the immediate-collection paradigm because a significant amount of axoplasm was removed before [35S]met was ever added to the incubation well. Artifactual leakage of radiolabelled proteins at the cannulae was less likely to occur in the immediate-collection paradigm because the MGA was cannulated prior to radiolabelling.

Preparation of tissue samples for analyses After the perfusion of an MGA, we injected mineral oil into the sheath to prevent the sheath from collapsing and to make the sheath easier to visualize for dissection. The 2-cm oil-filled M G A sheath inside the incubation well was dissected with fine forceps by removing the surrounding VNC tissue. A sheath sample contained glia, the MGA axolemma, and some cortical axoplasm which was not washed away by perfusion (see Results). In some cases (n = 4) a perfused M G A sheath was not filled with mineral oil or dissected from the VNC. Rather, the entire VNC was fixed for electron microscopy using protocols reported previously 2'45. At the time of collection, each sample of M G A axoperfusate, M G A sheath, and EWS, was frozen at -20°C. In several cases, axoperfusates were obtained from both the left and right MGAs of the same VNC, but each sample was individually analyzed. At the time of analysis, each sample of M G A axoperfusate, MGA sheath, and EWS was lyophilized into a dry pellet and redissolved in 12/A of SDS-PAGE sample buffer ~9. This final sample volume was chosen for several reasons. Firstly, when equivalent aliquots of all 3 sample types were loaded on a gel, proteins in each sample type were routinely detected by silver stain. Secondly, since the redissolved axoperfusate and sheath samples were from the same 2-cm length of MGA, equivalent volumes of the two samples could be directly compared, i.e., a 2-/~1 volume of axoperfusate or sheath sample represented a 0.3-cm length of each sample and 17% of the proteins in each sample. Thirdly, proteins were difficult to consistently

(2) Immediate-collection paradigm: cannulate, begin to perfuse, and add radiolabel to EWS for 3 h while continuing to perfuse. In several radiolabelling experiments (n = 7), a 2-cm length of VNC was positioned in the incubation well containing unlabelled EWS and the M G A was immediately cannulated rostral and caudal to the well as previously described. The rostral and caudal segments of the VNC outside the incubation well were covered with 250/~1 aliquots of mineral oil in order to completely isolate the cannulae from the saline inside the well. The M G A was perfused with 1-2 /~1 of unlabelled intracellular saline for 1-2 min before the unlabelied EWS was replaced with 250 #1 of EWS containing [aSS]met. The 2-cm length of VNC was incubated in the radiolabelled EWS for 3 h, during which about 8/A of axoperfusate was collected from the M G A at 1-h intervals for a total of 25 kd. At the end of the 3-h incubation and perfusion, a 100-/fl aliquot was collected from the radiolabelled EWS. The delayed-collection paradigm was used in the majority of radiolabelling experiments because the delayed-collection paradigm was less traumatic than the immediate-collection paradigm. That is, the M G A was not cannulated in the delayed-collection paradigm until after the radiolabelling incubation. The delayed-collection paradigm also allowed more time for [35S]met incorporation than the immediate-collection paradigm. The immediate-collection par-

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2 cm Fig. 2. Schematic diagram of a crayfish VNC placed in a Sylgard-fiUed petri dish having a small well surrounded by Vaseline. VNC ganglia labelled as follows: supraesophagea],thoracic (T1-Ts) and abdominal (ArA6). The severance site for chronic anucleate MGAs is shown in the left drcumesophageal connective. MGA cannulation and peffusion is accomplished by a collection cannula placed in the rostral, circumesophageal region of the MGA an(] an injection cannula placed approximately 2 cm away in the caudal, mid-abdominal region of the MGA. Note that the cannu|ac are placed in the MGA outside the Vaseline we|! used to contain radio|abelled EWS. Boxed area: enlargement to show an MGA injection cannula filled with intracellular saline and secured in the axon with a silk ligature.

71 detect in EWS samples which were concentrated less than 8x with respect to the original 100-/~1 EWS aliquot. In contrast, proteins were easily detected in axoperfusate samples which were only concentrated about 2x with respect to an original 25-/A aliquot.

Biochemical analyses of MGAs Total (radiolabetled and unradiolabelled) proteins were analyzed by SDS-PAGE and detected by silver stain. Aliquots (2/A) of the 12-/zl samples of MGA axoperfusate, MGA sheath or EWS were applied to 0.75-mm-thick, 7.5-17.5% gradient minigels and electrophoresed at 150 V for approximately 1 h. Molecular weight standards were run in parallel with all experimental samples. The gels were fixed in 50% methanol/10% acetic acid and silver-stained according to the method of Merril et al.27. Radiolabelled proteins were analyzed by SDS-PAGE and detected by fluorography. Aliquots (10/A) of samples analyzed by fluorography were taken from the same 12-/A samples as the 2-~1 aliquots analyzed by silver staining. 14C-labelledmolecular weight standards were run in parallel with all radiolabelled samples. The gels were fixed in 50% methanol/10% acetic acid, fluorographed with En3Hance, and dried at 60°C for 2 h. The dried gels were placed next to Kodak X-OMAT AR film in a -70°C freezer for 1-3 weeks. In some cases, fluorographed gels were rehydrated and silver-stained. The developed fluorographs and silver stained gels were scanned with a Hoefer GS 300 densitometer. To analyze the amount of total radioactivity (incorporated and unincorporated amino adds) by liquid scintillation counting (LSC), we added a 1-/tl aliquot from a 12-/zl sample (e.g. axoperfusate, MGA sheath, etc.) directly to 5 ml of 3a70 Complete Counting Cocktail. To analyze the amount of radiolabelled proteins by LSC, we spotted a 5-/zl aliquot of a sample onto filter paper and precipitated proteins with 10% trichloroacetic acid (TCA) according to the method of Mans and Novelli26. All samples were counted for 1 rain with a Beckman LSC-1800 counter. Counts in each aliquot were multiplied by the appropriate factor to obtain the counts in an entire 12-/~1sample. Unradiolabelled tissue was analyzed for immunoreactive levels of actin and fl-tubulin. Samples of axoperfusate, sheath, EWS, and VNC supernatant were obtained from the same preparation. The axoperfusate, sheath, and EWS samples were lyophilized and redissolved in 12-#1 of sample buffer, as described above. VNC tissue which remained after the MGA sheath dissection was homogenized with a micro-tissue grinder in 40/zl of riffs buffer (pH 7.4) for approximately 50 strokes, and centrifuged at 15,000x g for 5 min. The VNC supernatant was diluted 1:1 with SDS-PAGE sample buffer for a total volume of 80/A. An aliquot (12/fl) of each sample was loaded on a separate lane of one SDS gel. Proteins were transferred from an SDS gel to nitrocellulose in a Hoefer minigel transfer unit filled with transfer buffer containing SDS and methanol32. Rainbow molecular weight markers were used to monitor transfer efficiency. Each nitrocellulose sheet was air-dried, blocked with dehydrated non-fat milk powder in a sealable plastic pouch and incubated with the primary antibody, mouse monoclonal anti-fl-tubulin (1:500), for 90 min. The nitrocellulose was then incubated with the secondary antibody, goat anti-mouse IgG conjugated to colloidal gold (1:25). The entire procedure was repeated using mouse monoclonal anti-actin (1:500) as the primary antibody. Immunoreactive levels of actin and fl-tubulin in axoperfusate, sheath and EWS samples were given numerical values relative to the immunoreactive levels of VNC supernatants, which were assigned a value of 4. Two colleagues performed double-blind rankings by viewing unlabelled immunoblots. The amount of each sample type used for the rankings was as follows: aliquots of axoperfusate and sheath samples contained 100% (12/A) of the proteins from the same 2 cm length of a perfused MGA; the EWS aliquot contained 40% (100/tl redissolved in 12/tl/250/A of original EWS) of the proteins from the entire EWS; the VNC aliquot contained 15% (12/A/80/~1) of the proteins from the VNC supernatant.

RESULTS Samples of axoplasm were collected from crayfish M G A s by perfusion with intracellular saline. Compared to axoplasm in the squid giant axon 1'11 crayfish M G A axoplasm was fluid, possibly due to the low density of microtubules in the M G A axoplasmic core relative to other axons 45 and/or the complete absence of neurofilaments in crayfish 24'34. The fluidity of M G A axoplasm did not change when a calcium chelator ( E G T A ) or a protease inhibitor (leupeptin) was added to the intracellular saline.

Protein maintenance in intact and chronic anucleate MGAs Many axoperfusate protein samples (n = 50) were analyzed by S D S - P A G E and silver-stained. The b a n d i n g pattern of silver-stained axoperfusate proteins was very reproducible (Fig. 3A). Axopeffusate samples contained 20-30 silver-stained bands which were more intensely stained at 70, 55, 43, 38, 27, and 25 k D a (Fig. 3A: lines at right). Axoperfusate proteins obtained by a 3-h perfusion (n = 15) had the same b a n d i n g pattern as those obtained by a 10-15- min perfusion (n = 35), although the intensity of silver stain was usually greater for axoperfusate proteins obtained by a 3-h perfusion. Most importantly, the intensity and b a n d i n g pattern of silverstained proteins were the same in axoperfusate samples from intact M G A s (n = 35) and chronic anucleate M G A s (n = 15) allowed to survive for up to 6 months (Fig. 3A). Levels of actin and fl-tubulin immunoreactivity were compared for samples of M G A axoperfusate, M G A sheath, EWS, and V N C supernatant. VNC supernatant samples always stained very intensely for antibodies to actin and fl-tubulin at 43 k D a and 55 kDa, respectively. Immunoreactive levels in all other sample types were ranked with respect to the immunoreactive levels in VNC supernatants, which were assigned a numerical value of 4. Sheath samples stained more intensely for antibodies to actin and fl-tubulin than their respective axoperfusate samples from the same 2-cm length of M G A (Fig. 3B). (Sheath samples contained some axoplasmic proteins associated with the M G A axolemma.) Axoperfusate samples contained more actin and fl-tubulin than the more concentrated E W S samples. Most importantly, immunoreactive levels of actin and fl-tubulin were similar for samples obtained from intact M G A s and chronic anucleate M G A s severed up to 6 weeks (Fig. 3B: solid columns and hatched columns, respectively). These data suggested that actin and fl-tubulin were m a i n t a i n e d in chronic anucleate M G A s for at least 6 weeks.

72

Silver

(a)

Fig. 3. A: comparison of silver-stained proteins from intact and chronic anucleate MGAs. In this and subsequent figures, gel lanes labelled with asterisks indicate chronic anucleate MGAs and gel lanes without asterisks indicate intact MGAs. Numbers (200, 116, 97, 66 and 43) to the left of the gel lanes designate positions of molecular weight standards. Axoperfusates collected from an intact MGA (lane 1 ) and from a chronic anucleate MGA severed for 6 months (lane 2*) were electrophoresed on the same gel. Lines to the right of lane 2* indicate the most heavily silver-stained bands (70, 55, 43, 38, 27 and 25 kDa) in axoperfusate samples. The 200 kDa band in lane 2* was not consistently detected in axoperfusates from intact or chronic anucleate MGAs. Axoperfusates were collected by perfusing 25/zl of intracellular saline through 2-cm lengths of axons for 1.5-3 h and 17% of the proteins in each sample were loaded on the gel. B: comparison of levels of immunoreactivity to fl-tubulin and actin antibodies in samples of MGA axoperfusate (AX), MGA sheath (SH), and EWS. Levels of immunoreactivity (y-axis) were determined for each sample type (x-axis) from chronic anucleate (hatched columns) and intact (solid columns) MGAs. Top histogram: antibody = anti-fl-tubulin; bottom histogram: antibody = anti-actin. Unlabelled AX, SH and EWS samples were ranked for immunoreactive levels (4 > 3 > 2 > 1 > 0) relative to the immunoreactive level of VNC supernatant taken from the same preparation. Columns represent the mean immunoreactive level for each sample group of size: AX*, n = 7; AX, n = 8; SH*, n = 3; SH, n = 3; EWS*, n = 6; EWS, n = 4. The error bars above each column represent the standard error of the mean.

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Newly synthesized proteins in intact and chronic anucleate MGAs W h e n [35S]met was placed in the E W S surrounding a 2-cm length of V N C (Fig. 2), as many as 20-30 radiolabelled proteins were found in M G A axoperfusate samples (n = 31) obtained by the delayed-collection paradigm. The banding p a t t e r n of radiolabelled axoperfusate proteins analyzed by S D S - P A G E and fiuorography was very reproducible and always contained a heavily radiolabelled band at 46 k D a (Fig. 4A: arrow). Radiolabelled bands were also found at 96, 91, 74, 60, 44, 34, and 30 k D a in most axoperfusates (Fig. 4A: lines).

The banding pattern of radiolabelled axoperfusate proteins was the same for intact M G A s (n = 19) and chronic anucleate M G A s (n = 12) severed up to 9 weeks (Fig. 4A). This equivalence was particularly obvious when an axoperfusate sample from a s h a m - o p e r a t e d M G A was analyzed on the same gel with an axoperfusate sample from a chronic anucleate M G A taken from the same crayfish (n = 7, e.g. Fig. 4A: 1 and 2*). Radiolabelled axoperfusate proteins from chronic anucleate M G A s at shorter (1-3 weeks) postoperative times (Fig. 4A: 2*, 7*) were the same as those at longer ( 5 - 9 weeks) postoperative times (Fig. 4A: 5*, 8*, 9*, 10"). In addition, no obvious differences in the intensity of axoperfusate radiolabelling were noted between intact and chronic anucleate M G A s . Samples of axoperfusate and sheath samples from the same 2-cm length of M G A were c o m p a r e d on the same SDS gel (n = 2 M G A s ) or on different SDS gels (n = 14 M G A s ) . Since M G A sheath samples were always m o r e heavily radiolabelled than M G A axoperfusate sampies, fluorographed sheath samples (Fig. 4B: SH) were exposed to X-ray film for 1 w e e k whereas fluorographed axoperfusate samples (Fig. 4B: A X ) were exposed to X-ray film for 3 weeks. The banding patterns of radiolabelled proteins from axoperfusates and sheaths were similar. In such comparisons, heavily radiolabelled bands c o m m o n to axoperfusates and sheaths were a p p a r e n t at 60, 46, 44, and 34 k D a (Fig. 4B: lines). H o w e v e r , M G A sheaths had a heavily radiolabelled band at about 200

73

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kDa (Fig. 4B: solid arrow) which was not apparent in MGA axoperfusates. Furthermore, axoperfusate samples had a heavily radiolabelled band at 30 kDa (Fig. 4B: open arrow) which was not apparent in sheath samples. The radiolabelled 200 kDa protein in the sheath was apparently not transferred to the axoplasm. The radiolabelled 30 kDa protein in the axoperfusate could have been synthesized in the sheath and selectively transferred to the axoplasm. Alternatively, the 30 kDa protein could have been modified after being transferred to the axoplasm. These data suggested that many proteins synthesized in the glial sheath were transferred to intact and chronic anucleate MGAs, but that such transfer was somewhat selective. When radiolabelled and silver-stained proteins from the same axoperfusate sample could be directly compared on the same gel (n = 5) or on different gels (n = 10; Fig. 4C), two heavily radiolabelled proteins corresponded to two heavily silver-stained proteins (Fig. 4C: arrows). However, the majority of radiolabeUed axoperfusate proteins did not appear to correspond to silver stained axoperfusate proteins. We cannot assess exactly how many transferred (radiolabelled) proteins may correspond to axoplasmic (silver-stained) proteins because of the difficulty in comparing radiolabelled and silverstained proteins. This comparison is difficult due to dif-

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4 h after the start of [35 S]met incubations. Action potentials generated in the M G A segment outside the incubation well were conducted to the M G A segment inside the incubation well. The average action potential amplitude was 110 mV + 10 at t = 0 and 113 mV + 12 at t > 4 h. The average conduction velocity of action potentials through the incubation well was 11.5 rn/s at t = 0 a n d 1 0 . 4 m / s a t t_> 4 h . The integrity of the M G A axolemma was also examined in electron micrographs of perfused MGAs. MGAs perfused for 0.1-1 h (n = 4) were examined in crosssections and longitudinal sections taken at various places between the two cannulae. These electron micrographs (Fig. 5) showed that perfusion did not obviously disrupt the axolemma (Table III: #4). In fact, perfusion did not remove all the vesicles and microtubules from the cortical region just beneath the axolemma. Similar data have

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Perfused

Fig. 5. A: electron micrograph showing cross section of a control (non-severed, non-perfused) MGA. AX indicates axoplasm; SH indicates glial sheath. B: electron micrograph showing cross-section of an MGA which was perfused with intracellular saline for 10 rain. Bar = 1 ~m for A and B.

77 been reported for extruded or perfused squid giant axons 1'11'21. In contrast, when MGAs were peffused with a non-physiological saline containing fast green (n = 7), obvious disruptions of the MGA axolemma were observed 36. A set of other observations also indicated that the MGA axolemma was not disrupted during perfusions and/or that no leakage occurred at the cannulae tips. Firstly, the amount of axoperfusate collected was always less than or equal to the amount of intracellular saline that was injected (Table III: #5). Secondly, when radiolabelled axoperfusate proteins were collected by the delayed-collection paradigm, 1 to 8-/A perfusions collected a comparable amount of radiolabelled protein as did 25 /~1 perfusions (Table III: #6). Thirdly, when an additional 2/~l of axoperfusate was rapidly (5 min) obtained from a previously perfused MGA, TCA precipitable radioactivity was at background levels (Table III: #7). Fourthly, radiolabelled axoperfusate proteins collected by the more traumatic immediate-collection paradigm were comparable to those collected by the less traumatic delayed-collection paradigm (Table III: #8). Finally (Table III: #9), the ratio of TCA precipitable proteins in axoperfusates to that in sheaths (A/S = 0.1) was the same as that previously reported in an autoradiographic study which did not use the technique of axonal perfusion (see ref. 29: Tables I and II). DISCUSSION In this study we analyze axoplasmic proteins collected by perfusion to provide data on two phenomena that are possibly related: (1) the maintenance of axoplasmic proteins during LTS of chronic anucleate MGAs and (2) the appearance of newly synthesized proteins in the axoplasm of intact and chronic anucleate MGAs. Glial-axonal protein transfer could contribute newly synthesized proteins to intact and anucleate MGAs, thereby helping to maintain axoplasmic proteins necessary during the LTS of chronic anucleate MGAs.

Maintenance of axoplasmic proteins in chronic anucleate MGAs Previously published data have documented LTS of chronic anucleate MGAs by morphological and electrophysiological criteria 2'4'5'46. In this biochemical study we find that a wide variety of proteins, including actin and fl-tubulin, are maintained in anucleate MGAs for up to 6 months. The maintenance of proteins in a severed MGA for up to 6 months contrasts dramatically with the process of Wallerian degeneration in which axonal proteins are rapidly degraded within days to weeks in mammalian axons. The continued presence of many proteins

in the axoplasm of chronic anucleate MGAs must result from some combination of slow protein turnover, axoplasmic protein synthesis and/or intercellular transfer of newly synthesized proteins from adjacent cells such as glia.

Sources of radiolabelled proteins in axoperfusates The appearance of radiolabelled proteins in axoperfusates collected from MGAs isolated from their cell bodies is evidence for protein synthesis within, or external to, the axon. (Axonal transport of newly synthesized proteins from the MGA cell body 1°'43 is eliminated by axonal severance and by a Vaseline barrier to isolate the cell body from [35S]met.) Radiolabelled proteins could appear within axoplasm by axoplasmic processes such as: axoplasmic protein synthesis 12A3'18, posttranslational modification of existing axoplasmic proteins 16'~7, or mitochondrial protein synthesis. Our data suggest that axoplasmic processes contribute few, if any, newly synthesized proteins to the radiolabelled proteins collected in axoperfusates. For example, the immediate disruption of MGA axoplasm in the immediate-collection paradigm does not inhibit the appearance of radiolabelled axoperfusate proteins. In addition, only a few faintly radiolabelled proteins appear in axoperfusates when MGAs are perfused internally with intracellular saline containing laSS]met using our IN paradigm or when [aSS]met is incubated with axoperfusates in vitro for up to 5 h using our IV paradigm. When axoperfusates are obtained by the IN or IV paradigms, the faint radiolabelled banding pattern of the proteins is different from the heavily radiolabelled banding pattern of proteins in axopeffusates obtained by the delayed- or immediate-collection paradigms. Finally, the appearance of radiolabelled axoperfusate proteins obtained by the delayed-collection paradigm can be greatly reduced by placing an inhibitor of eukaryotic protein synthesis in the EWS, suggesting that radiolabelled axoplasmic proteins do not arise from posttranslational protein modification or mitochondrial protein synthesis. Radiolabelled proteins could also appear within axoperfusates because proteins synthesized external to the axon are somehow acquired by the MGA. Many observations or control experiments argue against artifactual leakage of radiolabeUed proteins from the EWS or the MGA sheath into the axoperfusate at the cannulae tips or across a disrupted axolemma (see Table III). We suggest that adjacent glia are the external source for most newly synthesized proteins detected in axoperfusates. The MGA glial sheath contains alternating layers of glia and collagen similar to that described for the squid giant axon and other unmyelinated invertebrate axons. The innermost (adaxonal) glial layer is separated from the

78 MGA axolemma by an extracellular space of only 20-80 nm 2'28. After severing the MGA, the glial sheath hypertrophies and glial cells often contain increased amounts of rough endoplasmic reticulum, perhaps reflecting an increased capability for protein synthesis 2. The crayfish MGA and surrounding glia have been reported to exchange various substances. For example, when low molecular weight dyes (Lucifer yellow CH, fast green, acridine orange and ethidium bromide) are iontophoresed or pressure injected into the MGA, the dyes rapidly appear in the surrounding glia36'44. HRP or BSA are also found in glia after injecting these tracer proteins into the M G A 14'45. In addition, HRP or BSA are taken up by the MGA after the protein is placed in the extracellular environment 45. Various observations suggest that glial-axonal protein transfer occurs for intact and chronic anucleate MGAs in crayfish3'28'29 and for intact giant axons in squid 11'2°22,39 Analyses of crayfish axopeffusates (see Results) and squid axoplasmic extrusions 2°-22'39 show that many newly synthesized proteins which have a broad range of molecular weights appear in the axoplasm of axons isolated from the cell body. In both crayfish and squid, the radiolabeUed banding patterns of axoplasmic and sheath proteins have many similarities. In both crayfish (see Results) and squid 2°-22'39 there is no complete identity between radiolabelled banding patterns of axoplasmic and sheath proteins, possibly reflecting a selective transfer of proteins from the sheath to the axoplasm. As discussed in detail for squid giant axons 11'21'22'38' 39, physiological mechanisms for glial-axonal protein transfer could include direct communication between glia and axon, exocytosis-pinocytosis, phagocytosis, or posttranslational protein translocation across membranes. Although there has been a morphological report of 'pores' between the crayfish MGA and adaxonal glia33, this observation has not been seen in other ultrastructural studies 2'36'45 and is not consistent with electrophysiological studies on MGAs and adjacent glia635'25. In squid, the removal of calcium from the extracellular saline significantly reduces the appearance of radiolabelled proteins in extruded axoplasm without affecting protein synthesis in the sheath, as would be expected if exocytosis-pinocytosis were a mechanism of glial-axonal protein transfer 21. However, in crayfish the removal of calcium from the radiolabelled EWS inhibits protein synthesis in the entire VNC (Sheller, personal observaton). In squid, the report that actin is transferred from the glia to the axon 39 is not consistent with exocytosispinocytosis as a mechanism for glial-axonal protein transfer because actin is not considered to be a secretory protein. Transfer of a cytoskeletal protein such as

actin could occur by axonal phagocytosis of adaxonal glia22'38'39 or possibly by direct posttranslational protein translocation across the glial plasmalemma.

Glial-axonal protein transfer and maintenance of axoplasmic proteins To maintain axoplasmic proteins, chronic anucleate MGAs exhibiting LTS must retain existing proteins (lack of protein degradation) and/or obtain newly synthesized proteins (local protein synthesis by a source other than the cell body). In this study, we present evidence that chronic anucleate MGAs do indeed receive newly synthesized proteins from a source other than the cell body (i.e. glial-axonal protein transfer). Since we find no qualitative or quantitative differences between radiolabelled axopeffusate proteins from intact and chronic anucleate MGAs, these data suggest that glial-axonal protein transfer may be equally important for the maintenance of axoplasmic proteins in intact and chronic anucleate MGAs. Although intact MGAs obtain proteins from the cell body by axonal transport 1°'43, some slowly transported proteins may take months to reach the most distal end of an MGA 23. Mechanisms for local maintenance of axoplasmic proteins such as glial-axonal protein transfer could provide an intact MGA with a supply of proteins more rapidly than axonal transport from the cell body. However, glial-axonal protein transfer or other mechanisms responsible for LTS of chronic anucleate MGAs cannot completely substitute for the MGA cell body, because chronic anucleate MGAs eventually degenerate 250-300 days after lesioning 2'4'5'46. Glial-axonal protein transfer could contribute to the maintenance of axoplasmic proteins in an MGA by supplying at least some of the axoplasmic proteins required by an MGA. Glial-axonal protein transfer could also contribute to the maintenance of axoplasmic proteins in an MGA by supplying proteins which extend the turnover times of a wide variety of axoplasmic proteins. For example, some of the newly synthesized proteins in crayfish MGAs might be HSPs transferred from glia, as reported for giant axons in squid 38'4°. Proteins of the HSP 70 family have been reported to function in protein translocation across membranes of endoplasmic reticulum and mitochondria s'9'31 and to remove clathrin from coated vesicles 7'35'41. HSPs may also function to stabilize proteins by protecting them from denaturation 3°. If this latter function is also seen in crayfish, LTS of chronic anucleate MGAs could result from a combination of mechanisms: intercellular transfer of glial-synthesized proteins to replenish axoplasmic proteins and intercellular transfer of glial-synthesized HSPs to stabilize existing axoplasmic proteins.

79 Acknowledgements. We wish to thank Mart Ballinger for help with electron microscopy, Henry Cobb for help with densitometry, Mellissa Made Curie Cobb for help with sample preparation, and Dr. William Mandy for consultations and use of laboratory space. We especiaUy thank Mark Smyers for invaluable assistance with

various biochemical procedures. This work was supported by a Texas Advanced Technology Grant to G.D. Bittner and a National Science Foundation Grant ECS 8915178 to G.D. Bittner (Co Principal Investigator).

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