Reduced protein synthesis in spinal anterior horn neurons in wobbler mouse mutant

EXPERIMENTAL

Reduced

NEUROLOGY

67, 423-432 (1980)

Protein Synthesis in Spinal Anterior Neurons in Wobbler Mouse Mutant

Horn

T. MURAKAMI, F. L. MASTACLIA. AND W. G. BRADLEY'

We studied protein synthesis in cervical anterior horn neurons in the “wobbler” mouse by measuring L-[4,5-:‘H]leucine incorporation using a light microscopic autoradiographic technique. Cytoplasmic incorporation of the isotope was significantly reduced not only in vacuolated nerve cells but also in those which were microscopically normal suggesting that impaired protein synthetic activity precedes the earliest morphologic changes seen with the light microscope. Isotope incorporation was reduced in small as well as large neurons suggesting that the disease process is not confined to a-motoneurons.

INTRODUCTION The “wobbler” mouse mutant (wr/wr) suffers from a recessively inherited degeneration of motoneurons (8) and has been considered a possible model for human motor neuron disease (3). The earliest change seen in spinal anterior horn neurons with the light microscope is a distinctive vacuolation of the neuronal perikaryon (9) which electron microscopic studies showed to be due to dilatation of the endoplasmic reticulum and Golgi apparatus ( 1). Only a small proportion of neurons in the anterior horn show this change and histologically normal nerve cells are also seen at all ages (9). The nature of the underlying metabolic disturbance which leads to neuronal degeneration in the wobbler is not known. ’ The work was supported by grants from the Medical Research Council, the Muscular Dystrophy Group of Great Britian, and the National Fund for Research into Crippling Diseases. The authors acknowledge the technical assistance of Miss M. Jenkison and Mr. N. Henderson and the helpful advice provided by Dr. K. Jones. Dr. Murakami’s present address is the Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyoku, Tokyo 113, Japan. Please address all correspondence to Professor Mastaglia. Professor W. G. Bradley’s address is Department of Neurology. Tufts-New England Medical Center, Boston. 423 0014-4886/80/020423-10$02.00/O Copyright All rights

D 1980 by Academic Press, Inc. of reproduction in any form reserved.

424

MURAKAMI,

MASTAGLIA.

AND

BRADLEY

To determine if protein synthetic activity is impaired in wobbler neurons we studied the incorporation of L-[4,5-“Hlleucine using a light microscopic autoradiographic technique. Our findings indicate a derangement of protein synthesis not only in vacuolated nerve cells but also in neurons which are not recognizably diseased at the light microscope level. MATERIALS

AND METHODS

Six pairs of 2-month-old male wobbler mice and their normal littermates were used in the study. Each animal was given a dose of 10 &i L-[4,5-3H]leucine (specific activity 40 to 53 Ci/mmol; The Radiochemical Centre, Amersham) by intraperitoneal injection between 1000 and 1200 h. Two pairs of animals were killed 0.75, 3, and 24 h after injection of the isotope by intracardiac perfusion of a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PH 7.4) for approximately 10 min, anesthesia having been induced beforehand by the intraperitoneal injection of Nembutal (60 mg/kg). After completion of the perfusion a wide cervical laminectomy was carried out quickly and after a further 2-h period of immersion in the same solution the cervical cord was carefully freed by dividing the spinal roots and removed. Having identified the upper four cervical dorsal roots the cord was divided transversely at the level of the fourth cervical segment and a transverse block of the whole cord was taken at this level and postfixed in the paraformaldehyde solution overnight. After washing in 0.1 M phosphate buffer four times and dehydration in graded solutions of alcohol, blocks were embedded in small plastic capsules filled with glycol methacrylate (12) which were then left overnight in a desiccator which had been evacuated and filled with nitrogen gas. Sections 2 pm in thickness were then cut using a Reichert OmD microtome and glass knives and placed on gelatin-coated glass slides. It is known that plastic tissue sections of this type show only a small variance in thickness (18). Under an Ilford IS0 906 safe light, tissue sections were covered with Ilford K2 emulsion maintained at 43°C in a water bath. After drying at room temperature overnight in complete darkness, sections were exposed in a black air-tight box which was kept 8 weeks in a deep freezer at -25°C. The sections were then developed by immersion in Kodak D- 19 developer (72 g/liter) for 6 min at 2O”C, fixed 8 min in 30% sodium thiosulfate solution, rinsed 2 min in Kodak Hypoclean (26 g/liter) stained 2 min in a 0.05% toluidine blue solution in 0.01 M benzoate buffer (PH 4.1), dried at room temperature, and then mounted with DPX mountant. The numbers of silver grains in the cytoplasm of all neurons with the nucleolus in the plane of section in one anterior horn were counted using an

SPINAL

MOTONEURON

PROTEIN TABLE

Neuronal

Surface

Area

and Nuclear

Area

1

in “Wobbler”

Mice

and Normal

Wobbler

Area

(pm?

Normal littermates (N = 178)

425

SYNTHESIS

Normal neurons (N = 222)

Littermates”

mice Abnormal neurons (N = 13)

Surface

254.4 2 14.0 224.1 -t 12.5 668.7 f 108.7 ------ ___________p < 0.05 _________--_----____________________.--_-----___ p < 0.00~ ______----.----_- _________.......

Nuclear

84.7 zk 3.2 81.3 + 2.9 71.4 + 9.1 _________________p > 0.1 _________________ --------------------..-..-------p > 0.1 -.....______________ ---.---- _____

’ The values shown are the mean + SE areas in all neurons with the nucleolus in the plane of section in one anterior horn in two animals. P values were calculated using Student’s t test.

oil immersion objective (X 100). The surface area of these same neurons was measured by planimetry on x844 photographic prints. Nuclear area was calculated using the following formula: area = iruhl4, where a is the length of the longest axis of the organelle, and h the greatest width measured at right angles to that axis, these measurements being made under an oil immersion objective using a Nikon filar micrometer eyepiece. Cytoplasmic area was calculated by subtracting the nuclear area from the cell surface area and grain densities (countslpm2) were then calculated. Background counts were made at each of the four corners of the methacrylate section not occupied by the tissue section. The average background count per square micrometer was then calculated and subtracted to yield a corrected grain density for each nerve cell. RESULTS Neurons showing various degrees of vacuolation and loss of Nissl material were present in the anterior horn of all wobbler mice examined and accounted for 5.0 to 6.4% of neurons in the anterior horn. Nerve cells showing these changes were not found in normal littermates. The surface area of vacuolated nerve cells was significantly greater than that of the normal anterior horn neurons of littermates (P < 0.01) and nuclear area was smaller but not significantly different (Table 1). The surface area of histologically normal neurons was significantly smaller in the wobbler than

426

MURAKAMI,

MASTAGLIA,

AND

BRADLEY

FIG. 1. A-normal anterior horn neurons in an unaffected littermate showing distribution of silver grains in the nucleus and cytoplasm. Toluidine blue. x 1900. B-reduced numbers of cytoplasmic silver grains in a large vacuolated neuron (arrowheads) in the anterior horn of a 2month-old wobbler. Toluidine blue. x 1900.

SPINAL

MOTONEURON

PROTEIN TABLE

Cytoplasmic

Incorporation in “Wobbler”

2

of L-[4,5-:‘H]Leucine Mice and Normal Cytoplasmic

grain

into Anterior Littermates.”

density

(grains/l000 Wobbler

Time (h) 0.75

Normal littermates 124.5 +- 7.7

(62) 3

74.2 k 4.4 (68)

24

84.7 ? 5.7 (48)

427

SYNTHESIS

Normal neurons 88.8 k 5.3 (95) P < 0.001 62.2 + 4.3 (59) P > 0.05 28.7 k 2.6

(68) P i

Horn

Neurons

pm? mice Vacuolated neurons 42.7

i- 9.1 (51

21.0 k 3.8 (41 18.5 2 6.8 (4)

0.001

fl The values shown are mean 2 SE silver grain counts in all neurons with the nucleolus the plane of section in one anterior horn in two animals at various times after administration of the isotope. P values were calculated using Student’s t test. N in parentheses.

in

in normal littermates (P < 0.0.5), but nuclear areas were not significantly different (Table 1). As shown in Table 2 and Figs. 1 and 2. silver grain densities were markedly reduced in the vacuolated neurons in the wobbler mice at 0.75,3, and 24 h. Grain densities were also reduced in histologically normal neurons in the wobbler mice (P < 0.001 at 0.75 and 24 h; N.S. at 3 h) but less so than in vacuolated cells (Table 2, Fig. 2). We were interested to know whether or not incorporation of the isotope was reduced to the same extent in a-motoneurons, which are thought to be preferentially involved in the wobbler mouse (1,9), and in other neurons in the anterior horn. Although there are no available data on the respective sizes of different functional groups of neurons in the anterior horn of the spinal cord in the mouse, it is assumed mainly on the basis of data from the cat (5) that the majority of a-motoneurons are large and that the smaller neurons in the anterior horn represent y-motoneurons, Renshaw cells, and interneurons. We therefore chose arbitrarily to subdivide neurons into two groups, those with a surface area greater than 300 pm2, and those with a surface area less than 300 pm?, on the assumption that the majority of a-motoneurons would be included in the first group. As shown in Table 3 and Figs. 3 and 4, grain densities were reduced in histologically normal

428

MURAKAMI,

0

L

MASTAGLIA,

AND BRADLEY

1; II %

3

24 Time (hours)

FIG. 2. Silver grain densities (grains/l000 pm’) in histologically normal (0) and abnormal (m) anterior horn neurons in wobbler mice and in the neurons of normal littermates (0) 0.75.3, 24 h after administration of L-[4,5-YH]leucine. Each point represents the mean t SE density in all neurons with the nucleolus in the plane of section in one anterior horn in two animals. The number of cells is shown in parentheses; the standard error is also shown.

neurons in both groups, the differences being significant at 3 and 24 h for neurons more than 300 pm’, and at 0.75 and 24 h for neurons less than 300 pm2. Counts were markedly reduced in all vacuolated neurons, the majority (77%) of which were more than 300 pm” in size. DISCUSSION The major assumption made in the present study is that the rate of incorporation of the administered radioactive amino acid provides a true measure of protein synthesis in the nerve cell. One premise upon which this assumption is based is that the amino acid has equilibrated with the endogenous amino acid precursor pool in the body as a whole and in the spinal cord (2). Care was taken to avoid factors which might influence the size of the precursor amino acid pool such as variations in the time of feeding in relation to administration of the radioactive isotope (11). The other assumption is that the isotope is incorporated into newly synthesised proteins and not bound nonspecifically to existing proteins in the cell. Free amino acids are washed out from tissue sections during the course of processing and it was shown in previous studies that binding of labeled amino acids in newly formed proteins accounts for 91 to 97% of the radioactivity retained in histologic sections (7). This technique has therefore been regarded as a reliable method for studying the rate of protein

SPINAL

MOTONEURON

PROTEIN

TABLE Cytoplasmic

Incorporation

Surface

Area

>300

3

of t.-14.5..‘H]L.eucine pm2

and

~300

Cytoplasmic Neurons

> 300

into

the

in “Wobbler”

pm’ grain

density

Anterior Mice and

(grains/1000

Horn Normal

Neurons with Littermates”

c: 300

mice

Wobbler

pm” mice

Time

Normal

Normal

Vacuolated

Normal

Normal

Vacuolated

(h)

littermates

neurons

neurons

littermates

neurons

neurons

0.75

121.7

k

IO.1

85.1

t

17.5

39.1

(8)

(13)

k 10.9

125.6

(4)

+ 9.3 (49)

74.5

i-

5.4

(IS) 24

69.6

2

k

6.5

27.1

(13) P < 0.02 6.3

(21)

the

54.0

26.9

-t

k

3.0

2.X

(20) P < 0.001

18.S

?

isotope.

P values

were

calculated

6.8

(4)

using

-t 5.7 (SO)

96.3

Student’s

t

5.6

t

64.6

2 5.2

(46) P -> 0.10 8.2

(27)

I’ The values shown are mean +- SE silver grain counts plane of section in one anterior horn in two animals

of the

74.0

(2)

89.1,

56.7

(87) P cc 0.001

P > 0.0s 3

a

grn2) Neurons

pm’

Wobbler

429

SYNTHESIS

29.5

(I) 15.0

f

2 3.5

(48) P K 0.001

in all neurons with the nucleolus at various times after administration f test.

2.0

(2)

in

N in parentheses.

synthesis in nerve cells and in other tissues (7). We used buffered paraformaldehyde as a fixative in preference to glutaraldehyde which is known to cause polymerization of free amino acids in tissue sections (16). This study showed a significantly reduced incorporation of L3H]leucine into cervical anterior horn neurons in Z-month-old wobbler mice suggesting that protein synthesis is reduced in these cells. The significance of this finding is uncertain. Although the reduced [3H]1eucine incorporation in vacuolated neurons could simply be part of a severe overall metabolic derangement in such cells, the finding of a similar abnormality in histologically normal nerve cells suggests that impaired protein synthesis is an early manifestation of the metabolic disturbance in the wobbler neuron and one which is fairly closely related to the basic defect. In a separate study we found that the incorporation of [3H]uridine into the nucleus was reduced both in vacuolated and in histologically normal anterior horn neurons in the wobbler mouse (14) and that the RNA content of these cells was reduced (15). The reduced protein synthesis in these cells may therefore be secondary to impaired nuclear synthesis of messenger and/or ribosomal RNA.

430

MURAKAMI,

MASTAGLIA,

AND BRADLEY

140-

120-

loo-

r 8

fJo-

z 2 2

60-

40 t

FIG. 3. Silver grain densities in neurons with a surface area more than 300 pm2. Other details as for Fig. 2.

The possibility of excessively rapid degradation of newly formed proteins or transportation from the nerve cell body into the axon needs to be considered, but seems unlikely. Although an increased lysosomal enzyme content was found in the white matter of the cervical cord (lo), this is probably secondary to the myelinated nerve fiber degeneration which occurs and there is no definite evidence of increased catabolic activity in nerve cells in the spinal grey matter (10). Autoradiographic studies of

,

I 24

Time

Ihours)

FIG. 4. Silver grain densities in neurons with a surface arealess than 3OOpm*. Other details as for Figs. 2 and 3. x-three vacuolated nerve cells.

SPINAL

MOTONEURON

PROTEIN

SYNTHESIS

431

protein degradation in wobbler neurons were not made. Studies of axoplasmic flow showed no evidence of increased transport of materials out of the nerve cell. In one study it was suggested that the slow phase of intraaxonal flow was impaired in a subpopulation of neurons (3), whereas in another study no significant abnormalities of fast or slow axonal transport of protein were found (4). It has been implied in the past that the degenerative process in the wobbler mouse selectively involves motor neurons (1, 9), and that other functional groups of neurons in the anterior horn are not affected. The present findings point to a metabolic abnormality in small as well as large neurons in the anterior horn suggesting that y-motoneurons, interneurons, and Renshaw cells may be affected as well as a-motoneurons. In conclusion, our findings indicate that the genetic defect in the wobbler mouse is expressed in the majority of neurons in the anterior horn of the spinal cord, and that one of its early effects is a reduction in protein synthetic activity in the nerve cell which is probably secondary to impaired nuclear synthesis of RNA. REFERENCES I. ANDREWS, J. M. 1975. The fine structure of the cervical spinal cord, ventral root and brachial nerves in the wobbler (wr) mouse. J. Ne~ropnrh. Exp. Nelm)l. 34: 12-27. 2. BARONDES, S. H. 1976. Protein metabolism in the regulation of nervous system function. Pages 328-341 in G. J. SIEGEL PI al.. Eds.. Basic Neurochemisrry. Little, Brown, Boston. 3. BIRD, M. T., E. SHUTTLEWORTH. JR., A. KOESTNER, AND J. REINGLASS. 1971. The wobbler mouse mutant: an animal model of hereditary motor system disease. Acra Neuropath. 19: 39-50. 4. BRADLEY, W. G.. AND E. JAROS. 1973. Axoplasmic flow in axonal neuropathies. II. Axoplasmic flow in mice with motor neuron disease and muscular dystrophy. Bruin 96: 247-258. 5. CAMPA, J. F.. AND W. K. ENGEL. 1971. Histochemical and functional correlations in anterior horn neurons of the cat spinal cord. Science 171: 198-199. 6. DROZ, B., AND H. WARSHAWSKY. 1963. Reliability of the radioautoradiographic technique for the detection of newly synthesized pr0tein.J. Histochem. Cytochem. 11: 426-435. 7. DROZ, B., AND C. P. LEBLOND. 1963. Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography. J. Comp. Neural. 121: 325-337. 8. DUCHEN. L. W.. D. S. FALCONER, AND S. J. STRICH. 1966. Hereditary progressive neurogenic muscular atrophy in the mouse. J. Physiol. (London) 183: 53-55P. 9. DUCHEN, L. W., AND S. J. STRICH. 1968. An hereditary motor neurone disease with progressive denervation of muscle in the mouse. The mutant ‘wobbler’. J. Neural. Neurosurg. Psychiut. 31: 535-542. 10. HIRSCH, H. E.. J. M. A. ANDREWS, AND M. E. PARKS. 1974. Acid hydrolases and other enzymes in secondary demyelination: a quantitative histochemical study in the wobbler mouse. J. Neurochem. 23: 935-941.

432

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MASTAGLIA,

AND

BRADLEY

JAROS, E. 1977. Studies of the Peripheral Nervous System of the Dysrrophic Mouse. Ph.D. Thesis, University of Newcastle upon Tyne. 12. LEDUC. E. H.. AND W. BERNHARD. 1967. Recent modifications of the glycol methacrylate embedding procedure. J. Cilrrusrrucr. Res. 19: 196- 199. 13. MAHLER, H. R. 1976. Nucleic acid metabolism. Pages 342-361 in G. J. SIEGEL, et (II., Eds.. Basic Neurochemistry. Little, Brown. Company, Boston. 14. MURAKAMI, T.. F. L. MASTAGLIA, D. M. A. MANN. AND W. G. BRADLEY. 1978. A 11.

Quantitative Morphological und Microspectrophotome/ric Anterior Horn of the Wobbler Mouse. IVth International

Study

of Neurones

in the

Congress on Neuromuscular

Diseases, Montreal. September 1978 (Abstract 7). 15. MURAKAMI, T., F. L. MASTAGLIA, AND D. M. A. MANN. 1978. A microspectrophotometric study of neurones in the anterior horn of the wobbler mouse. Neuropufhol. Appl. Neurobiol. 5: 78 (abstract). 16. PETERS, T.. JR., AND C. A. ASHLEY. 1967. An artefact in radioautography due to binding of free amino acids to tissues by fixatives. J. Cell Biol. 33: 53-60. 17. RAPPOPORT. D. A., R. R. FRITZ. AND J. L. MYERS. 1969. Nucleicacids. Pages lOI- 119in A. LAJTHA, Ed., Handbook of Neurochemistry. Vol. I. Plenum. New York. 18. ROGER, A. W. 1973. Techniques mf Altrorudio~rcrplz~, 2nd ed. Elsevier. London/New York. 19. SCHULTZE. B. 1969. In A. W. POLLISTER. Ed., Physicul Techniqurs in Biologicui Reseurch. Vol. III, Port B: Autoradiography at the Cellular Level, 2nd ed. Academic Press, New York.