Axonal transport in mdx mouse sciatic nerve

Journal of the Neurological Sciences, 1989, 92:267-279

267

Elsevier JNS 03205

Axonal transport in mdx mouse sciatic nerve Shu-ichi Yamashita 1, Hitoshi Takenaka 2, Seiichiro Sugimoto 1, Etsuo Chihara 3, Atsushi Sawada 3, Shigeru Matsukura ~ and Minoru Hamada 2

Departments of JThird Internal Medicine, 2Hygiene, and 3Ophthalmology, Miyazaki Medical College, Miyazala 889-16 (Japan)

(Received 6 March, 1989) (Revised, received 14 April, 1989) (Accepted 17 April, 1989)

SUMMARY Anterograde and retrograde flows of acetylcholinesterase (ACHE) in sciatic nerves of adult m d x mice were compared with those of normal mice. Specific molecular forms of AChE were resolved by high-performance liquid chromatography such that slow anterograde (G 1 + G2) , fast anterograde and fast retrograde (G 4 and A12 ) flows could be simultaneously studied. Although we found no difference in the total AChE activity and the molecular forms in non-ligated nerves between m d x and the normal mice, ligated nerves showed significant differences. The total AChE activity accumulated at the proximal segment of ligated nerve was higher in m d x mice than in normal mice after 24 h ligation. The GI + G2 molecular forms were accumulated more in the proximal segment o f m d x than the normal. A12, on the other hand, was more abundant in both segments of m d x mice than the normal. No statistically significant difference in the accumulated amount of G 4 molecular form was present between m d x and the normal mice at either proximal or distal segment. These results indicated that axonal flow in sciatic nerve likely plays a role in muscle regeneration, and that the transport machinery in dystrophin-deficient m d x neuron is probably normal.

Key words: Acetylcholinesterase; Muscular dystrophy; m d x mouse; Axoplasmic transport; Muscle regeneration

Correspondence to: Dr. Minoru Hamada, Department of Hygiene, Miyazaki Medical College, Kiyotake-cho,Miyazaki-gun,Miyazaki 889-16, Japan. 0022-510X/89/$03.50 © 1989Elsevier Science Publishers B.V. (BiomedicalDivision)

268 INTRODUCTION The mdx mouse is a homozygous inbred strain of C57BL/10 mice isolated by Bulfield et al. (1984) which exhibits a histopathologically severe but clinically undetectable skeletal muscle myopathy. The muscle of mdx mice has been shown to lack "dystrophin" (Hoffman et al. 1987) and the genetic mutation causing the disease has been localized with the mouse homologue of the human Duchenne muscular dystrophy (DMD) gene (Ryder-Cook et al. 1988). As such, the marx mouse is considered as an animal model for human DMD. Despite the recent advances m understanding of the genetics and biochemistry of DMD. there are major unresolved questions concerning the pathogenesis of the disease. Specifically, why do dystrophin-deficient mice (mdx) and young CXMD dogs and DMD boys manifest active myopathy but no clinical weakness, while older CXMD dogs and DMD boys exhibit striking progressive weakness? The recent identification of dystrophin as being related to spectrin (Davison and Critchley 1988; Koenig et al. 1988) supported the implication of possible primary defects of neurons in the etiology ofDMD (Hoffman et al. 1988). We thus investigated axonal transport in the mdx mouse to examine possible involvement of neuronal defects in DMD. Acetylcholinesterase (ACHE) in mammals is composed of six molecular forms, identified as either globular molecules (G) or asymmetric molecules possessing a collagen-like tail (A). Both the G and A forms can oligomerize into G 1 (monomer), Gz (dimer), G 4 (tetramer), m 4 (tetramer), A 8 (octamer), and A tz (dodecamer) (Bon et at. 1979). Since Lubinska and Niemierko (1971) first reported axoplasmic transport of AChE in canine sciatic nerves, G~, G 2, G4, and A12 have been used as indicators to study the molecular mechanism of axoplasmic transport (Di Giamberardino and Couraud 1978; Couraud and Di Giamberardino 1980; Massouli6 and Bon 1982: Vitadello et al. 1983; Marini et al. 1986). G~ and G 2 are likely conveyed by slow anterograde transport, and G4 and A12 by fast anterograde and retrograde transport (Di Giamberardino and Couraud 1978; Couraud and Di Giamberardino 1980: Massouli6 and Bon 1982). Thus, it is possible to investigate all types of axonal flows simultaneously by examining axonal transport of individual AChE forms resolved by high-performance liquid chromatography (HPLC). The present study showed that transport ofG~ + G2 by the slow anterograde flow and of A12 by both of the fast anterograde and retrograde flows was significantly increased in the sciatic nerve ofmdx mice compared to controls. The G4 molecular form showed no such increase. Thus axonal transport in the mdx mouse is not obviously perturbed by dystrophin deficiency. Moreover, our results indicate that an active myopathy is reflected in the volume of axonal transport. MATERIALSAND METHODS Animals Mdx mice were generously provided by Dr. G. Bulfield through the Central Institute of Experimental Animals. Japan, and genetically matched C57BL/10 control

269 mice by the Japanese National Institute of Genetics. Both strains were given conventional diet and water ad libitum. All breeding was carried out by brother-sister mating.

Operative procedures After brief anesthesia with diethyl ether, animals (41-43 weeks old) were anesthetized further by intraperitoneal injection of pentobarbital (30 mg/kg). The left sciatic nerve was then exposed at the mid-thigh region, gently freed from surrounding connective tissues, and tied tightly with a silk thread (8-0 gauge). The right sciatic nerve was treated as the left nerve except that ligation was not performed. The incision was then closed with thread suture. Nerve exposure time did not exceed 2 min and the whole operation was carried out at room temperature while anesthetized within 5 min. Mice came out from the anesthesia no sooner than 30 min. After 24 h of ligation, mice were killed by overexposure to diethyl ether. Blood was withdrawn from the heart in order to eliminate possible contamination of blood-bome cholinesterase during preparation of the nerve samples. The segment of nerve on each side of ligation site (3 mm) was immediately removed for AChE testing. As the non-ligated control nerve was presumed to contain low AChE activity, a 5-mm nerve sample was removed from the right thigh for determination of the control level of total AChE and its molecular forms. The amount of protein in a Triton X- 100 extract of 5 mm sciatic nerve corresponded to about 54~o of the value which was expected from the protein yield per length calculated from the extract of 3 mm nerve in our method (unpublished data).

AChE assay Nerve segments were homogenized by a Branson Sonifier 250 at 15 W and 0.5-sec intervals for 14 cycles in 200 #1 of an ice-cold solution containing 1 M NaC1, 1~o Triton X-100, 1.9mg/ml ethyleneglycol-bis(/~-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA), and 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.0) with 1 mg/ml bacitracin and 0.16 mg/ml benzamidine as proteases inhibitor (extraction buffer). As membrane-bound AChE coexists with the soluble form, Triton X-100 at the micellar concentration is often used for their extraction (Di Giamberardino and Couraud 1978; Saku and Brimijoin 1978; Bon et al. 1979; Couraud and Di Giamberardino 1980; Massouli6 and Bon 1982; Sorensen et al. 1982; Vitadello et al. 1983; Marini et al. 1986). We examined the efficiency of various detergents to yield the maximal AChE activity from sciatic nerve (Fig. 1). The results suggested us to use 1.0~o Triton X-100 for the AChE extraction. Homogenates were immediately centrifuged at 20 000 x g for 20 min at 4 ° C. Twenty #1 of the resultant supernatant was provided for determination of the total AChE activity. Assay was carried out at 25 ° C according to the method of Ellman (1961) with a slight modification in an "assay medium" which included 1 M NaC1, 0.75 mM acetylthiocholine (AcThCh), 0.5 mM 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and 40 mM Hepes (pH 7.0) in the presence of 0.1 mM tetraisopropylpyrophosphoramide (iso-OMPA) as the inhibitor against pseudocholinesterase (Couraud and Di Giamberardino 1980; Marini et al. 1986). The amount of thiocholine released was determined by measuring the increase

270 lO E

8

E

~ 6 c [

f

4,

~

2q

x

00

10

2.0

Detergent

30

4.0

5 L

(%)

Fig. 1. AChE activity in 4-mm segment of SD rat sciatic nerve homogenate in extraction buffer containing various concentrations of detergents. Detergents used were Triton X-100 (O), CI2Es (A), CHAPS ( 0 ) , and n-oetyl-fl-glucoside (Fq). Reaction conditions were described in the text. AChE was solub[fized efficiently by either 1.0% Triton X-100 or 0.3% C~2Es.

in the absorbance at 412nm with a Cary 2290 spectrophotometer at 25 °C. A spontaneous hydrolysis of AcThCh was also estimated and was taken into account for calculation of the enzymatic activity of sciatic nerve samples. One unit AChE represents the quality of enzyme hydrolyzing 1 nmol AChE in 1 min.

HPLC analysis H P L C with Superose 6 (1 x 30 cm, Pharmacia, Uppsala, Sweden) was applied for fractionation of AChE molecular forms. Elution was carried out with the extraction buffer at a constant flow rate of 0.2 ml/min (Model 1359T Soft-Start Pump, Bio-Rad, California) at 4 °C. The AChE molecular forms were identified by their molecular weight. Thyroglobulin (Mr 670000), ]~-amylase (M r 200000), bovine serum albumin (M r 66 000), and cytochrome c ( M r 12 400) were used as standards to determine the molecular weight of AChE molecular forms. The elution volume of blue dextmn 2000 was regarded as the void volume. One tenth ml of the nerve extract was subjected to the column and 0.2-ml fraction was collected. All fractions were immediately mixed with 1 ml of the "assay medium', and the absorbance at 412 nm was determined (Beckman DU65 spectrophotometer) after an incubation for 15 h at 4 °C. The activity of each AChE form was estimated by calculating its relative proportion in the total activity applied to HPLC, assuming that loss in the AChE activity during H P L C was similar for all forms (Couraud and Di Giamberardino 1980; Marini et al. 1986). Others Protein concentration in the remaining nerve extract was determined using bicinchoninic acid (Smith et al. 1985), and bovine serum albumin was used as the standard. AcThCh, iso-OMPA, protease inhibitors, and the molecular weight standard proteins were purchased from Sigma Chemical Co. (St. Louis, MO) and bicinehoninic acid from Pierce Chemicals (Rockford, IL). Other reagents were purchased from either Wako Chemicals (Osaka, Japan) or Nakarai Chemicals (Kyoto, Japan). All reagents were of analytical grade, and Type-I reagent grade water was used throughout the study.

271 TG (670K)

Blue dext. (2000K) lO

AMY (200K) BSA (66K)

~ Cyt C (124K)

E 08 c cq

06 0.4 <

02 A12 00

0

~'~

.... 2O

40 Fraction number

60

80

Fig. 2. Molecular forms of AChE in SD rat sciatic nerve by gel filtration using HPLC. HPLC with Superose 6 separated four AChE molecular forms; A,2, G4, G 2, and G~. HPLC was thus efficient to examine the molecular forms of ACHE. Molecular weight was calibrated with standard proteins; blue dextran (Mr 2 000 kDa), thyroglobulin (670 kDa), E-amylase (200 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa).

Statistical analysis Statistical treatment of the AChE activities were made according to the unpaired Student's t-test.

RESULTS

HPLC patterns of AChE molecularforms from mdx and normal mice sciatic nerves The HPLC analysis confirmed the presence of 4 peaks (AI2, G4, G2, G1) in the SD rat sciatic nerve (Fig. 2), that had been detected by a centrifuge in a linear sucrose density gradient (5-20~, w/v) (Di Giamberardino and Couraud 1978). The HPLC profiles of AChE molecular forms in non-ligated sciatic nerves ofmdx mice were shown in Fig. 3. No significant difference was observed with the normal mice. Two peaks

0.2 E c

G4

o.1 J71

<

A12 0.0,

~

2O

40

60

80

Fraction number

Fig. 3. Molecular forms of AChE in non-ligated mdx sciatic nerve by gel filtration using HPLC. Gel filtration profile showed 3 major peaks, one of which was likely the combined peak of G1 and G 2. Relative amount of each molecular form was estimated as the area under the corresponding peak.

272 04

A

5

04 O~

~z

~0~

m

00~ 2O

4O Fraction number

60

80

40

60

80

04

E

B

tz

02

£

00'"

2[

Frachon number

Fig. 4. (A) Molecular forms of AChE in proximal segment (----i---) and distal segment ( ---) in ligated mdx sciatic nerve. (B) Molecular forms of AChE in proximal segment(---HI--) and distal segment ( -4 in ligated normal mouse sciatic nerve. Both proximal and distal segments included all 3 AChE molecular forms. AChE activity was higher at the proximal segment than the distal side for both species of mice.

corresponding to Al2 and G4 and a fused peak of G1 + G 2 were detected. Thecombined amount of G~ and G2 were used for estimation of low molecular weight forms (Vitadello et al. 1983; Marini et al. 1986), as both molecular forms were likely conveyed by a slow anterograde flow (Di Giamberardino and Couraud 1978; Couraud and Di Giamberardino 1980) and the sedimentation profile o f A C h E in dy/dy mice in the presence of sucrose density gradient (Brimijoin and Schreiber 1982) was similar to the H P L C pattern shown in this report. The area of each molecular form was estimated as described in the legend of Fig. 3. H P L C patterns of AChE molecular forms in the proximal and distal segments at the ligation o f m d x and normal mice sciatic nerves were compared in Fig. 4. All 3 peaks of AChE molecular forms were detected in the extracts of both proximal and distal segments. Proximal segments showed higher AChE activity than those of distal segments in both mdx and the normal mice. There was no a p p ~ e n t difference in the elution volume of each peak between nerves from rndx and normal mice. Axoplasmic transport of AChE in sciatic nerve of mdx mouse Axoplasmic transport of AChE is often discussed from the viewpoint of the AChE activity per length of nerve samples (Lubinska and Niemierko 1971; Di Giamberardino and Couraud 1978; Couraud and Di Giamberardino 1980; Brimijoin and Schreiber 1982; Vitad¢llo et al. 1983; Marini et al. 1986). However, as diseased animals, such as

273 diabetes mellitus and dy/dy mice, are generally smaller and lighter than the normal animals, their sciatic nerves may be thinner than that of normals. It may thus be inadequate to compare the properties of nerves between the diseased model and normal animals from the AChE activity per length of nerves. Furthermore, it is difficult to measure nerves precisely and extract enzymes with the same efficiency from nerves. In order to overcome these problems, we determined the protein concentration in the extract as well as the length of nerves. The value of protein-based AChE activities was about 7 times larger than that based on the length of samples in both of the proximal and distal segments for all molecular forms of mice (Tables 1-4). In contrast, the non-ligated samples showed about 11 times larger activities based on the protein than those based on the length. These discrepancy might be attributed to the difference in the length of samples provided for extraction, i.e., 3 mm for the ligated and 5 mm for the non-ligated nerves, as described in Materials and Methods. Comparison of the accumulated AChE activity, regardless per length or mg protein, should be made between corresponding segments of mdx and normal mice. There was no statistically significant difference in the total AChE activity between mdx and normal mice in the non-ligated (control) sciatic nerve (Table 1). M d x mice showed significantly higher AChE activity in the proximal segment than normal mice, while no difference in the accumulated activity of AChE was found in the distal segments between mdx and normal mice. Table 2 summarizes the content of A~2 molecular form in various nerve segments. There was no difference in the activity of the A~2 molecular form in non-ligated side between the mdx and normal mice. In both of the proximal and distal segments, mdx mice showed significantly higher activity of A~2 molecular form than normal mice. On the other hand, accumulation of the G 4 molecular form showed no statistically significant difference between the mdx and normal mice (Table 3). It was thus concluded that only A~2 delivered by fast axonal flow increased in sciatic nerves of mdx mice. No significant difference in the content of G~ + G 2 molecular forms in non-ligated nerves was found between the mdx and normal mice (Table 4). Accumulation of the G~ + G 2 molecular form increased in the proximal segments of mdx nerves, while there was no difference in the activity in the distal segments. These results indicated that the slow anterograde axonal flow increased in sciatic nerve of mdx mice.

DISCUSSION Since Di Giamberardino and Couraud (1978, 1980) have reported the fast anterograde and retrograde transport of A~2 and G 4, and the slow transport of G 2 and G~ in chick and rat sciatic nerves, the axoplasmic transport of AChE has been studied in dy/dy mice as well as diabetic mice (Vitadello et al. 1983; Marini et al. 1986). The major advantage of studying axoplasmic transport of AChE molecular forms is to estimate all types of axonal flows simultaneously. Altered axonal flow in muscular dystrophy has been reported in dy/dy mice (Jablecki and Brimijoin 1974; Komiya and Austin 1974; Tang et al. 1974; Brimijoin and Jablecki 1976; Brimijoin and Schreiber 1982), in which axoplasmic transport of transmitter related enzymes such as choline acetyltransferase

0.539 + 0.026 0.779 _+ 0,066 t

4.20 + 0.270 5.41 + 0.360*

0.279 +_ 0:230 0.415 + 0.065

1.85 +0.140 2,64 +_ 0.360

units/mg

0:252 _+ 0.016 0.234 _+ 0.013

units/mm

Control

Normal md~:

0.0391 + 0.0011 0.0563 ~ 0,0043 ~

0.274 + 0,011 0391 ± 0.024 ~

0.0184 _+ 0.0024 0.0278 y_ 0.0013 ~

units ,mm

units/ram

units/mg

Distal

Proximal

0.123 _~ 0.019 0.185 - 0.01 t ~

units/mg

0.0166 +_ 0.0028 0.0212 +_ 0.00t7

units~mm

Control

All results are presented as the mean + SEM ~n = 5): mdx mice show significantly more I~P < 0.01. *P < 0.05) AChE activity than normal mice,

AI2 AChE ACTIVITY

TABLE 2

Normal mdx

units/mm

units/mm

units/mg

Distal

Proximal

All results are presented as the mean + SEM (n = 5); mdx mice show significantly more (*P < 0,05) AChE activity than normal mice.

TOTAL AChE ACTIVITY

TABLE 1

0.179 ~ 0.038 0.239 T 0.018

units/mg

2.69 + 0.263 2.71 + 0.230

units/mg

4~

0.508 + 0.025 0.626 _+ 0.051

3.60 + 0.251 4.35 _+ 0.290

0.222 +_ 0.017 0.310 + 0.046

1.47 + 0.100 1.93 + 0.220

units/mg

0.209 _+ 0.016 0.182 + 0.0078

units/mm

Control

Normal mdx

0.0460 _+ 0.003 0.0965 + 0.020*

0.325 + 0.020 0.667 + 0.130'

0.0379 + 0.005 0.0830 + 0.029

units/mm

units/mm

units/mg

Distal

Proximal

0.251 + 0.031 0.522 + 0.170

units/mg

0.0242 + 0.004 0.0309 + 0.006

units/mm

Control

All results are presented as the mean + SEM (n = 5); m d x mice show significantly more (*P < 0.05) AChE activity than normal mice.

G, + G 2 AChE ACTIVITY

TABLE 4

Normal mdx

units/mm

units/mm

units/mg

Distal

Proximal

All results are presented as the mean _+ SEM (n = 5); there is no significant difference in AChE activity between mdx and normal mice.

G 4 AChE ACTIVITY

TABLE 3

0.262 + 0.053 0.364 + 0.082

units/mg

2.22 + 0.240 2.11 + 0.150

units/mg

276 (Jablecki and Brimijoin 1974), dopamine-fl-hydroxylase (Brimijoln and Jablecki 1976) and AChE (Brimijoin and Schreiber 1982) are considerably decreased. However. the dy/dy mouse is considered a primary neuropathy (Bradley and Jenkison t975: Carnwath and Shotton 1987). Thus, the alterations of axonal flow might be attributed to defects in the nerve tissue. Recent studies have suggested primary expression of dystrophin m neurons (Hoffman et al. 1988) and have indicated sequence homologies between dystrophin and spectrin (Davison and Critchley 1988; Koenig et at. 1988), a protein thought to be of structural importance for axons. We thought it important to measure axoplasmic transport of AChE and its molecular forms in mdx sciatic nerves, and thereby study a possible relationship between abnormalities of axonal transport and dystrophin deficiency. There was no statistically significant difference in the total AChE and molecular form distribution in the non-ligated portion of sciatic nerves between mdx and normal mice. independent of whether the activities of nerve extracts were expressed per unit length or per mg of protein (Tables 1-4). Given this normal base fine, it was then feasible to compare the functions of sciatic nerves of mdx mice with the normal mice by estimating the activities of the total AChE and molecular forms after tigation of sciatic nerves. However, the AChE activities showed significant differences in values depending on whether values were normalized for unit length or protein content (Tables 1-4). Those discrepancies were likely due to the amount of nerve isolated for extraction. Although homogenization by sonication ensured the constant extraction of proteins, the result indicated that yield of the AChE activity significantly depended upon the size of nerve samples. Therefore, the discussion should be limited on comparison of the accumulated activity of AChE between mdx and the normal mice only at the corresponding segments. Hoffman et al. (1988) recently reported that mdx mouse lacks of dystrophin not only in muscles but also in neurons. Dystrophin deficiency was expected to perturb the axonal flow in sciatic nerve of mdx mouse. The present study demonstrated that transport of GI + G 2 by the slow anterograde flow and of A~2 molecular form by the fast antero- and retrograde flows were significantly increased in the sciatm nerve of mdx mice compared to normal mice (Tables 2 and 4). This fact indicated that unlike in the dy/dy mouse (Brimijoin and Schreiber 1982) or diabetic animals (Marini et at. 1986' Vitadello et at. 1983) dystrophin deficiency at least did not decrease axonal flow of AChE in the sciatic nerve of the mdx mouse. Despite that the mdx mouse has been considered as an excellent model for human D M D (Hoffman et al. 1987; Ryder-Cook et al. 1988), dissimilarities have been pointed out between mdx mice and human D M D patients in their behavior and life span (Bulfield et at. 1984; Dangain and Vrbovfi 1984; Carnwath and Shotton 1987). Histopathological studies, for instance, revealed that the skeletal muscles of adult mdx mice were composed of regenerated fibers of normal size with internal nuclei and scattered small foci of active degeneration and regeneration (Bulfield et al. 1984: Camwath and Shotton 1987). Dangain and Vrbov~t (1984) reported that none of the tension output, speeds of contraction and relaxation, or weight of tibialis anterior muscles was different between adult mdx and normal mice of the C57BL/6J strain. These indicate that mdx

277 mouse is a model animal suitable not only for human DMD but also for study on the degeneration-regeneration of muscle fibers (Dangain and Vrbovh 1984; Carnwath and Shotton 1987). We thus attempted to examine possible correlation between axonal flow and degeneration and regeneration of muscles. Three types of axonal flows with different directions and rates have been identified by a study of AChE transport: the fast anterograde, slow anterograde and fast retrograde flows, while slow retrograde flow has yet not been demonstrated. Among various forms of ACHE, Ax2 and G 4 forms are transported by the fast anterograde and retrograde flows, and G~ + G 2 forms by the slow anterograde flow. The role of fast anterograde flow, which carries smooth endoplasmic reticulum (SER) with most membrane-bound molecules such as AChE (Couraud and Di Giamberardino 1980), is considered to participate in supporting synaptic functions at the nerve end (Vale 1987). The G4 molecular form has been shown to leave nerve terminals in response to nerve stimulation (Saku and Brimijoin 1978). On the other hand, the A~2 molecular form is exclusively found in the region of skeletal muscle containing neuromuscular junction, appears by innervation, and disappears after denervation (Hall 1973; McLaughlin and Bosmann 1976; Vigny et al. 1976a, b; Koenig and Vigny 1978; Sketelj et al. 1978). The AI2 molecular form is therefore a useful marker of the neuromuscular junction and the stage of innervation. The selective increase in anterograde A~2 flow reasonably represents enhanced innervation caused by active muscular regeneration (Table 2). The present study showed increment in the amount of A12 transported in mdx mice, whereas there was no difference in the G4 accumulation between mdx and normal mice (Table 3). This fact indicated that active muscular reinnervation proceeds in the regenerated muscle of the mdx mice. The slow anterograde flow is assumed to participate in maintenance of the axonal structure and the transport ofcytoskeletal proteins and soluble substances (Vale 1987). Increased slow anterograde flow shown by GI + G2 molecular forms in mdx sciatic nerve might also influence active muscular regeneration o f m d x mice. Several candidates have been listed as neuronal trophic factors for muscle growth, which include sciatin in chick sciatic nerve (Markelonis et al. 1980, 1982), insulin-like growth factor I (Hansson et al. 1987), and AChE (Couraud and Di Giamberardino 1980). However, substantially effective neuronal trophic factor(s) is hereafter to be identified, and such factors influencing muscular regeneration of mdx mice have yet not been found. The increased transport of the G~ + G 2 forms in mdx mice may indicate a possible involvement of substances driven by the slow anterograde flow in muscular regeneration (Table 4). Fast retrograde axonal flow carries endoplasmic reticulum vesicles most of which are lysosomal vesicles (Vale 1987), so that retrograde flow may play roles in disposing mechanisms, such as transporting endocytotic vesicles and wastes in synapses towards the cell body. The retrograde flow, moreover, is likely functioning as a messenger from the nerve end to the cell body. Increase in retrograde transport of the A I2 molecular form in mdx sciatic nerve suggested that retrograde axonal flow would transmit the informations of muscle necrosis, which implied the anterograde axoplasmic transport of neuronal trophic factor for muscle regeneration.

278 ACKNOWLEDGEMENTS

We thank Dr. M. Saito of the Central Institute of Experimental Animals (Japan) and Dr. K. Sakakibara of the Japanese National Institute of Genetics for their kind supply of mdx and C57BL/10 mice, respectively. We also thank Dr. K. Tsuchiya of Miyazaki Medical College for his guidance and suggestions on carrying out animal experiments. We thank Dr. E. P. Hoffman of Children's Hospital, Harvard Medical School, for his valuable suggestions and criticism in completing this manuscript. This study was supported by grant No. 63570837 from the Ministry of Education, Science and Culture of Japan.

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