the effects of ciliary neurotrophic factor on motor dysfunction in wobbler mouse motor neuron...

ORIGINAL ARTICLES

The Effects of Ciliary Neurotrophc Factor on Motor Dysfunction in Wobbler Mouse

Motor Neuron Disease Hiroshi Mitsumoto, MD,"? Ken Ikeda, MD," Tomas Holmlund, MD," Tom Greene, PhD,$

Jesse M. Cedarbaum, MD,O Vivien Wong, PhD,O and Ronald M. Lindsay, PhD$

~ ~~

Ciliary neurotrophic factor is the first neurotrophic factor to show survival-promoting effects in developing motor neurons in vitro, in ovo, and in vivo. In the present study we tested the effects of recombinant rat or human ciliary neurotrophic factor in the wobbler mouse model of motor neuron disease. Mice received 1 mg/kg of the factor or a vehicle solution subcutaneously three times a week for 4 weeks, after the disease was diagnosed between the ages of 3 and 4 weeks. Although treatment with rat ciliary neurotrophic factor (n = 6) resulted in delayed weight gain ( p < 0.001), grip strength normalized to body weight in the factor-treated mice was significantly greater ( p < 0.02) and declined at a slower rate ( p < 0.05) compared to that in vehicle-treated animals. Human ciliary neurotrophic factor (n = 27) produced no change in body weight and reduced paw position and walking pattern abnormalities ( p < 0.001 and p < 0.02, respectively). After 4 weeks of treatment, the mean grip strength of human ciliary neurotrophic factor-treated animals was twice as great ( p < 0.001) and declined at a much slower rate ( p < 0.005) than that of control mice. The time required to run 2.5 f t was less ( p < 0.005) and muscle twitch tension was greater ( p < 0.002) in ciliary neurotrophic factor-treated animals. Thus, ciliary neurotrophic factor retarded the disease progression and improved muscle strength in this motor neuron disease model.

Mitsumoto H, Ikeda K, Holmlund T, Greene T, Cedarbaum JM, Wong V, Lindsay RM. The effects of ciliary neurotrophic factor on motor dysfunction in wobbler mouse

motor neuron disease. Ann Neurol 1994;36:142- 148

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease of undetermined cause. Although a recent discovery of mutations in the gene responsible for superoxide dismutase in some patients with familial ALS has increased our understanding of possible disease mechanisms 111, there is still no effective treatment for this disorder. Several hypotheses may explain the cause of ALS 12-41; one suggests that a lack of trophic substance causes neuronal degeneration 141. However, only recently have neurotrophic factors be- come available in sufficient quantities (via recombinant DNA technology) to permit testing of their therapeutic potential in motor neuron diseases (MNDs) f5-71.

Ciliary neurotrophic factor (CNTF) was originally identified as a survival factor for chick ciliary gan- glion neurons 181. CNTF is found predominantly in Schwann cells of peripheral nerves, and supports the survival of a variety of neurons and glial cells [6,9- 1 11. CNTF was the first neurotrophic factor to show survival-promoting effects in developing motor neurons in vitro 112-141, in ovo 1151, and in vivo 116, 171.

The recent study by Masu and colleagues 1181 revealed that mice lacking the CNTF gene develop motor neuron depletion and its corresponding weakness only as adults, suggesting that CNTF may function to maintain mature motor neurons. We therefore sought to determine whether CNTF shows similar supportive effects on diseased motor neurons in a naturally occurring MND.

The wobbler mouse is an extensively studied animal model of MND {19-231. Affected mice develop an autosomal, recessively inherited, lower MND that predominantly affects the forelimbs. A pathognomonic perikaryal vacuolar degeneration occurs in lower motor neurons, resulting in loss of cervical motor neurons and denervation muscle atrophy [21,24, 251. The diag- nosis is made as early as the ages of 3 to 4 weeks, and the ensuing progressive motor dysfunction is character- ized by weakness in the front paws and forelimb, gait disturbance, and eventual flexion contractures of the forelimb muscles. The reliability of the clinical diagno- sis at this stage has been confirmed repeatedly by previ-

From the Departments of *Neurology, ?Neuroscience, and fBiosta- tistics and Epidemiology, The Cleveland Clinic Foundation, Cleve- land, OH, and IRegeneron Pharmaceuticals, Tarrytown, NY. Received Jan 4, 1994, and in revised form Feb 14. Accepted for publication Feb 16, 1994.

Address correspondence to Dr Mitsumoto, Neuromuscular Section, Department of Neurology, S91, Cleveland Clinic, 9500 Euclid Ave- nue, Cleveland, OH 44195.

142 Copyright 0 1094 by the American Neurological Association

ous histological findings [2 1, 231. The disease progression is most rapid in the first few weeks after onset and then appears to slow [21, 251. We now report the effects of treatment with recombinant rat and human CNTF on motor dysfunction in this MND model. Pre- liminary accounts of these studies have appeared in abstract form 126-28).

Materials and Methods Wobbler mice used in this study came from the wobbler mouse colony of The Cleveland Clinic Foundation [23]. The investigation was approved by the Animal Research Commit- tee of The Cleveland Clinic Foundation. In the initial pilot study with recombinant rat CNTF, 18 wobbler mice were studied. In the second experiments with human CNTF, 56 wobbler mice were tested. Power calculations based on results of the first study with rat CNTF indicated that a sample size of 28 mice per group would be necessary to detect a significant change in grip strength without body weight adjustment.

Purified recombinant rat or human CNTF (Regeneron Pharmaceuticals) was prepared in aliquots at 500 p,g/ml[7]. At the time of clinical onset of the disease, a subcutaneous injection of CNTF at 1.0 mgikg (or a vehicle solution) was given under halothane anesthesia, and thereafter repeated three times a week for the next 4 weeks. The dose was chosen based on previous studies with Mnd mice (291. The investigators were masked to the treatment (either CNTF or vehicle solution) of the mice during the study.

Semiquantitative Assessments All semiquantitative and quantitative assessments [30] were performed when the disease was diagnosed and then weekly thereafter for 4 weeks immediately before CNTF administration. Paw position was graded as depicted in Figure 1. Walk- ing pattern was graded as follows: 0 = normal; 1 = trembling; 2 = wobbling; 3 = curled paw walking; and 4 = jaw walking (in which the jaw is used to support the body instead of the paws).

Quantitative Assessments Animals were weighed weekly. Grip strength of the front paws was measured at the point when the mouse released a horizontal wire grasped by both front paws as a result of gentle traction applied to the tail. The wire was connected to a gram dynamometer. The maximal grip strength of five trials was recorded. When paw contractures developed and animals were no longer able to grip the wire, grip strength was recorded as 0 gm E30). Running time was defined as

Grade0 Grade1 Grade 2 Grade3 Grade 4 ~

Fig I . Grading for paw position: 0 = normal; 1 = atrophied paw; 2 = curled digits; 3 = carled wrists; and 4 = forelimb fiexion contracture.

the shortest time to run 2.5 ft [30}, and the best of three trials was recorded. Open field locomotion was recorded by a video camera mounted in an examination cage in which a 2-in square grid was drawn on the floor. The recording was begun 2 minutes after the mice were transferred to the cage. The number of squares traversed in 9 minutes was counted.

In Vivo and In Vitro Muscle Twitch Tension At the end of the 4-week treatment period with human CNTF, 22 animals were anesthetized with halothane, and twitch tension of the biceps muscle produced by direct elec- trical stimulation of the musculocutaneous nerve was measured according to the methods described by Ikeda and Mit- sumoto E31). The maximum force generated by five serial biceps muscle contractions was recorded. The musculocutaneous nerve was then transected to establish that the electri- cal stimulation triggered no further muscle contraction. After the procedure, the biceps muscle was dissected and weighed. Twenty-one additional mice were anesthetized with halothane, and the entire biceps and triceps muscles were re- moved. The muscle tissue was maintained in a tissue bath containing Krebs-Ringer's solution at 37"C, and aerated with a 95% oxygen-5% carbon dioxide gas mixture. Supramaxi- ma1 stimulation was applied for 2 msec at a frequency of 0.2 Hz, using a Grass S44 stimulator with flanking platinum plate electrodes, and the maximal twitch tension was recorded [3 1, 32). After the procedures, the muscle tissue was dried and weighed.

Statistical Analyses The effects of CNTF treatment on semiquantitative variables were tested by applying the nonparametric Wilcoxon rank-sum test. Quantitative variables were analyzed using repeated-measures analysis of variance. Rates of change were assessed from the slopes of least-squares regression lines computed for each mouse using the baseline and the four weekly follow-up measurements, while the total change from baseline to week 4 was assessed by the simple differences between these two measurements. The regression slopes and the total changes were compared between treatment groups using unpaired t tests and the Wilcoxon rank-sum test. The results were consistent for each method; consequently, only the t test results are reported. These analyses were conducted both with and without weight adjustment. The differences between the two groups in quantitative measurements ob- tained at a single follow-up time point (e.g., the muscle twitch tensions) were analyzed using the unpaired t test. All tests were two-sided; (Y was set at 0.05.

Results Recombinant Rat Ciliary Neurotrophic Factor In the first experiment we studied the effects of recombinant rat CNTF or a vehicle in 18 wobbler mice. Two from each group died in the first week of treatment and were excluded from the analyses. All the mice gained weight during the study, and although the rat CNTF-treated mice (n = 6) initially lost weight, after the first week their rate of weight gain was similar t o that in control mice (n = 8). The differences in body

Mitsumoto et al: Clinical Effects of CNTF 143

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Fig 2. Mean (+ SEM) grip strength in vehicle- and rat (A) or human (B) cilia y neurotrophic factor (CNTF)-treated wobbler mice. (A) Grip strength normalized t o body weight to account for weight loss with rat CNTF treatment. At week 4, body weight-adjnsted grip strength was signz$cantly greater (* p < 0.02) in rut CNTF-treated mice (n = 6) than in control mice In = 8). (B) Grip strength in human CNTF-treated wobbler mice was greater at week 2 , 3 , and 4 (n = 27Igroup; "p < 0.03: **p < 0.003; and "*"p < 0.001).

weight at and after the first week of treatment between rat CNTF and vehicle solution were significant ( p < 0,001). By 4 weeks, control animals had gained an average of 4 gm, whereas rat CNTF-treated mice had gained 2 gm.

Paw position abnormalities developed less rapidly in treated mice ( p < 0.05), but there was no difference in walking pattern. Grip strength, although on average greater in CNTF-treated animals, did not differ statistically between the two groups. However, the overall mean weight-ad justed grip strength was significantly greater for the rat CNTF-treated group than the vehicle-treated group at the end of treatment ( p < 0.02) (Fig 2). Although the disease progressed in both groups of animals, the rate of decline in adjusted grip strength was significantly slower in the CNTF group than in the control group ( p < 0.05). Rat CNTF treatment slowed the decline in both actual and weight- adjusted running time more than did the control treatment, but the differences were not statistically significant.

Reliability of Quantitative Measurements Before the study with human CNTF, the reliability of quantitative measurements was tested. In 10 untreated wobbler mice the means of the maximum percent devi- ations over three measurements taken approximately 1 hour apart (computed as 100 x [highest - lowest)/ [mean of three replicated measurements}) were 1 1.1% at baseline and 3.396 at week 4. The means of the

Vehicle

Human CNTF

, Secondweek , , Fourth Week , 1 2 3 4 1 2 3 4

Grade

Fig 3. Efects of human ciliary neurotrophic factor (CNTF) treatment on the progression of paw position abnormalities in wobbler mice with motor neuron disease. Each column represents the percentage of animals scored at each grade at each week. Between-group differences (n = 271gronp) were statisticalb signifcant at week 4 (p < 0.001).

maximum percent deviation over repeat running time measurements were 2.6% at baseline and 2.6% at week 4. These quantitative measurements were thus reliable.

Recombinant Human Cilia y Neurotrophic Factor The second study focused on human CNTF and employed 28 mice per group. At baseline, the two groups of wobbler mice did not differ in semiquantitative and quantitative assessments. One control and 1 human CNTF-treated animal died during the first week of the experiment.

In contrast to rat CNTF, human CNTF administered at the same dose produced no weight loss. Figure 3 summarizes the semiquantitative assessment of paw position abnormalities. At baseline, all wobbler mice were scored as grade 1. However, at the end of treat-

144 Annals of Neurology Vol 36 No 2 August 1994

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F i g 4. Mean (? SEM) time taken for wobbler mice to run 2.5 f t . Running time increases as motor function decreases. At the end of treatment, cilia y neurotrophic factor (CNTFbtreated animals were almost twice as fast as vehicle-treated mice ("p < 0.005i (92 = 27lgroup).

ment, the majority of human CNTF-treated wobbler mice had not progressed beyond grades 1 and 2, whereas the majority of vehicle-treated animals had deteriorated to grades 3 and 4 ( p < 0.001). CNTF treatment also resulted in similar findings with walking pattern abnormahties ( p < 0.02).

During the first week of the study, average grip strength rapidly declined from baseline in both control and CNTF-treated groups (see Fig 2). However, differences emerged after the second week of treatment, so that by the end of treatment (4 weeks), the grip strength was on average twice as great in CNTF-treated animals ( p < 0.001). The reduction in grip strength from baseline to 4 weeks was significantly slower in human CNTF-treated wobbler mice than in vehicle- treated animals ( p < 0.005). When grip strength was adjusted by body weight, the differences were still significant (data not shown) ( p < 0.02).

Running time (Fig 4 ) increased over time in both control and CNTF-treated groups of wobbler mice. However, the rate of the increase was significantly slower in human CNTF-treated animals from baseline to the fourth week ( p < 0.001). At the end of treatment, the running time of the human CNTF-treated mice was approximately half that of vehicle-treated animals ( p < 0.005). In contrast, open field locomotion scores diminished to a similar extent over 4 weeks in both groups of animals, suggesting that the positive effect of human CNTF on running time was not the result of a general stimulation of locomotor activity.

Muscle twitch tensions in vehicle- and human

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Fig 5. In vivo (A) and in vitro (B) twitch tension in wobbler mice after 4 wee& of treatment with ciliay neurotrophic factor (CNTF) or a vehicle solution. Horizontal marks indicate mean muscle twitch tension. CNTF treatment resulted in greater twitch tension both in vivo (p < 0.001) and in vitro (p < 0.002).

CNTF-treated animals are shown in Figure 5 . In in vivo studies, the muscle of human CNTF-treated wobbler mice generated approximately 5 7 % more tension (4.00 * 0.76 gm) than did that in vehicle-treated mice (2.55 * 0.83 gm) ( p < 0.001). In vitro biceps- triceps muscle twitch tensions were also significantly (77%) higher with human CNTF (3.63 * 0.99 gm) than with vehicle treatment (2.05 2 0.91 gm) (j < 0.002). Twitch tensions adjusted for muscle weight were also higher in human CNTF-treated mice in both tests ( p < 0.02).

Discussion Assessment of the rate of progression of motor dysfunction, in both animals and humans, is not a straight- forward task. Lang and colleagues C33j and Kozachuk and coworkers { 30 j developed semiquantitative functional grading scales to assess the change of motor dysfunction in wobbler mouse MND. The present study clearly showed that systemic CNTF administration significantly delayed the worsening of paw position and walking pattern, although the disease continued to progress. To evaluate more objectively the course of motor dysfunction and muscle strength, we employed quantitative measurement techniques, such as grip strength and running time [30]. For all quantitative

Mitsumoto et al: Clinical Effects o f CNTF 145

measures used in the present study, we carefully analyzed the reliability of these measurements and found that the measurement techniques were reproducible. Therefore we believe that the differences in the results found between CNTF and vehicle treatments are reliable.

Grip strength proved to be the key quantitative test in the first study using rat CNTF, but the results were statistically significant only when the grip strength was adjusted to body weight. At the dose used, rat CNTF impaired weight gain in wobbler mice, whereas the same dose of human CNTF did not. This difference may be explained by the fact that despite an 85% pro- tein sequence identity, it is now clear that human CNTF is four to five times less potent than rat CNTF 171. Panayotatos and colleagues {34] found that this difference in potency resides at a single amino acid residue. Taken together, these observations suggest that the efficacy of the rat CNTF used in the first study was partially compromised by weight loss and perhaps poor nutritional status. Thus, a reduction in rat CNTF dosage may have resulted in an efficacy similar to that of human CNTF without impairing weight gain.

The observation that CNTF treatment can reduce the rate of loss of grip strength and running speed in wobbler mice was supported by measurements of in vivo and in vitro muscle twitch tension. The in vivo twitch tension examines the functional integrity of the motor unit, including the motor neuron, neuromuscular junction, and skeletal muscle, whereas in vitro twitch tension directly measures muscle contractility. Although muscle twitch tensions rapidly increased over time in normal litter mates, they steadily decreased with age in wobbler mice, reflecting the physiological effect of motor neuron degeneration on muscle [31]. Although muscle weight was approximately 20%

higher in CNTF-treated than in control animals {28], differences in muscle weight alone cannot account for the increase in twitch tensions because the difference remained significant even after normalization to muscle weight.

The mechanism of the beneficial action of CNTF in wobbler MND may be both myotrophic and neurotrophic, as it is now clear that the CNTF receptor is expressed in skeletal muscles as well as in motor neurons 19, 351. Helgren and coworkers {361 showed that denervation-induced muscle atrophy in the adult rat is markedly attenuated by CNTF treatment, with con- comitant preservation of contractile properties. Fur- thermore, Forger and coauthors I1 71 reported that CNTF prevents naturally occurring motor neuron loss and associated muscle atrophy in developing rats.

Nonetheless, it is unlikely that the improvement of motor dysfunction associated with human CNTF resulted solely from a myotrophic action of CNTF, because our histometric studies in wobbler mice indicated that CNTF treatment seemed to have preserved motor neurons and their axons {28]. Systemically administered CNTF appears to access motor neurons via re- ceptors located on their axon terminals, which lie out- side the blood-brain barrier. Curtis and associates 1371 recently determined that CNTF is transported from the periphery to motor neuron cell bodies by receptor- mediated retrograde axonal transport. Furthermore, DiStefano and colleagues (Regeneron Pharmaceuticals, personal communication, 1994) found rapid activation of the immediate early gene tzs-1 1 in the ventral spinal cord after subcutaneous injection of CNTF, indicating a direct action of CNTF on motor neurons.

The effects of CNTF have been investigated in two other murine MNDs {29, 38). The Table summarizes the differences among murine MNDs, including the

SummaT of Murine Motor Neuron Diseases and the EHectJ of Ciliavy Neurotrophic Factor (CNTF) Treatment

Motor Neuron Progressive Motor Degeneration (Mnd) Neuronopathy (Pmn)

Features Wobbler Mouse Mouse Mouse

Inheritance Age at onset Paralysis Life expectancy Denervation CNTFIroute Benefits Functional scalesb Quantitative tests Histologyb Reference

AR 3-4 wk Forelimb 6 mo Yes Subcutaneous Yes Yes Yes Yes 28 and the present study

AD 5-11 mo Hindlimb Not affected Late Subcutaneous Yes N o Yes No 29

AR 3 wk Hindlimb 6-7 wk Yes Intraperitoneala Yes N o N o Yes 38

"Intraperitoneal injection of mouse D3 tumor cells transfected with a CNTF genomic DNA construct. bTechniques used to demonstrate the effects of CNTF. AR = autosomal recessive; AD = autosomal dominant.


wobbler mouse, and the techniques used to test the effects of CNTF treatment. The three murine MNDs differ in inheritance pattern and clinical features. Be- cause CNTF improved motor dysfunction in all three of these MNDs, the therapeutic action of exogenous CNTF may not be restricted to a single MND; rather, CNTF may enhance the protective mechanisms by which cells compensate for a variety of injury and disease insults. Clinical studies to determine the long- term safety and clinical benefits of recombinant human CNTF in patients with ALS are underway 1391.

Drs Cedarbaum, Lindsay, and Wong are full-time employees and shareholders in Regeneron Pharmaceuticals. We gratefully acknowl- edge the generous support of the ALS Association and donations from ALS patients and their families.

Mrs Gisela Bunge provided excellent technical assistance. Prof Ma- sao Kinoshita and Dr Yasuo Iwasaki, Toho University School of Medicine, gave their encouragement and support to Dr Ikeda while he stayed in Cleveland. Mr Tom Lang, CCF Medical Editing Services, kindly reviewed our manuscript.

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