0006-Ch10.pdf

Canine Motor Neuron

Disease: A View from

the Motor Unit

Martin J. Pinter, Timothy C. Cope, Linda C. Cork,

Sherril L. Green, and Mark M. Rich

CONTENTS

10.1 Introduction

10.2 Human Motor Neuron Disease

10.3 Understanding the Progression of Motor Neuron Disease

10.4 Hereditary Canine Spinal Muscular Atrophy

10.5 Motor Unit Functional Properties in HCSMA

10.5.1 Younger Homozygotes

10.5.2 Older Homozygotes

10.5.2.1 Failing Motor Units

10.5.2.2 Effects of 4-Aminopyridine

10.5.3 Neuromuscular Transmission in HCSMA

10.5.4 Mechanisms Responsible for Tetanic Failure in HCSMA

10.5.4.1 Neuromuscular Degeneration

10.5.4.2 Axonal Abnormalities

10.6 The Pathological Process Underlying HCSMA

10.6.1 Oxidative Stress

10.6.2 The Role of Activity

10.7 Summary

References

10.1 INTRODUCTION

The motor neuron diseases are a collection of progressive, neurodegenerative dis-

orders. No effective treatments exist for these disorders primarily because underlying

pathological mechanisms are poorly understood. The need for a greater understand-

ing of possible mechanisms has led to the use of several animal models of motor

neuron disease. This chapter considers recent work on one of these models called

Hereditary Canine Spinal Muscular Atrophy (HCSMA), ﬁrst identiﬁed by Cork and

colleagues. 1,2 General problems and issues associated with human motor neuron

disease are considered ﬁrst, followed by a review of recent work in HCSMA that

focuses on the functional properties of motor units and how their function is lost

during disease progression.

10.2 HUMAN MOTOR NEURON DISEASE

The most common form of motor neuron disease in humans is amyotrophic lateral

sclerosis (ALS), or Lou Gehrig’s disease, named after the famous baseball player.

Most cases of ALS (85 to 90%) appear without a history of family involvement and

are termed sporadic while the remainder are inherited and are called familial ALS

(FALS). Although ALS is viewed by many as the prototypical motor neuron disease,

upper motor neuron involvement (corticospinal) is considered to be a cardinal feature

of the disease. In general, exclusive involvement of motor neuron degeneration

among the motor neuron diseases appears to be rare. 3 Thus, most cases of ALS do

not feature exclusive motor neuron degeneration and so are not disorders of a speciﬁc

neuronal cell type. It is clear, however, that motor neurons are particularly vulnerable,

and understanding this vulnerability remains a central focus of research effort.

An intriguing feature of ALS is how the onset of the disorder varies. Approxi-

mately 30% of the cases exhibit the ﬁrst signs of weakness focally among muscles

innervated by cranial motor neurons (bulbar onset), 20 to 30% of cases show an

onset in distal leg muscles, while about 30% show the ﬁrst signs in distal hand

muscles. 3 Considerable variance is noted in the balance between upper and lower

motor neuron signs as well. 3 The basis for this variability is not known, but one

possibility is that it is caused by different mechanisms. Other observations, however,

indicate that the differing onset versions all tend to regress to a state that features

common clinical signs if sufﬁcient time for full disease progression occurs. 3 These

observations are more compatible with common underlying mechanisms, while the

variability in the onset and character or rate of progression could indicate the

existence of modifying factors. Several modifying genes have been identiﬁed in one

animal version of motor neuron disease. 4 The identiﬁcation of modifying factors,

whether genetic or environmental, could be of particular importance because they

could, in principle, be used to control at least the progression of the disorder.

One of the most insidious aspects of ALS is that the actual onset of the disease

process occurs well before the victim becomes aware of its existence. Available

estimates indicate that as much as 50% of the original innervation in a muscle is

lost before weakness is ﬁrst noted. 5,6 The mechanism underlying this phenomenon

is the well-known ability of surviving motor terminals to sprout and reinnervate

nearby denervated muscle ﬁbers. 7 Although this mechanism preserves muscle force,

it also creates uncertainty in distinguishing clinically between loss of innervation

by sprouted motor terminals vs. loss of original innervation. It is thus not surprising

that the mechanisms that cause the initial denervation of muscle and loss of motor

unit function in ALS are not well understood. An understanding of these mechanisms

seems particularly necessary because their operation leads to loss of motor unit

function, the single most necessary problem in motor neuron disease.

Classically, the role of motor neuron cell death in the loss of motor unit function

has received the most emphasis. In large part, this is due to the relative ease with

which motor neuron cell death can be demonstrated with routine histological exam-

ination of autopsy material. Human autopsy results, however, are dominated by

disease endstage phenomena, and while there is no doubt that cell death explains

the permanent loss of motor units and paralysis of ALS, it remains uncertain whether

cell death actually accounts for the initial loss of motor unit function. The clinical

electrophysiological tests used to diagnose ALS depend on the motor neuron’s ability

to activate muscle ﬁbers 8,9 and can only detect that denervation has occurred. These

methods cannot distinguish between functional denervation that occurs because motor

neurons are dysfunctional and denervation that occurs because motor neurons have died.

10.3 UNDERSTANDING THE PROGRESSION

OF MOTOR NEURON DISEASE

These considerations underscore the potential importance of understanding events

that occur before motor neuron cell death. In the clinical literature, there are reports

suggesting that a preliminary phase of motor unit dysfunction occurs in ALS patients.

Several investigators have reported abnormal decrement of motor unit potentials

during repetitive activation, 8,10–12 and in vitro studies indicate that quantal content is

decreased at ALS motor terminals. 13 Some of these ﬁndings may reﬂect the func-

tional properties of immature nerve endings belonging to terminal sprouts, but they

also raise the possibility that motor unit functional failure precedes motor neuron cell

death. Understanding the mechanisms that underlie motor unit failure is particularly

important because this failure is the foundation of weakness in motor neuron disease.

Technical issues and other problems limit the ability to obtain from human

studies a detailed understanding of how motor units fail in motor neuron disease.

Fortunately, a number of animal models of motor neuron disease are available for

study. A common concern, however, is that none of these models exactly replicate

the human disorder. This concern is partly justiﬁed because all these models are in

non-primate species which lack the direct (monosynaptic) corticospinal projections

to motor neurons that degenerate in human disease. Nevertheless, these animal

models provide unique opportunities to test ideas about mechanisms that can cause

motor neuron degeneration and dysfunction but cannot be explored at all in human

disease. Animal models have been especially instructive regarding certain forms of

hereditary motor neuron disease and for expanding our understanding of possible

molecular mechanisms. The lack of exact replication of the human disease is perhaps

best viewed as an important constraint that must be considered when generalizing

results obtained from these models to the human disease.

10.4 HEREDITARY CANINE SPINAL

MUSCULAR ATROPHY

In order to examine mechanism issues in motor neuron disease, we have in recent

years studied an animal model called Hereditary Canine Spinal Muscular Atrophy

FIGURE 10.1 Muscle locations and grades of spontaneous electromygraphic (EMG) activity

in an HCSMA homozygote. Spontaneous EMG activity recorded in resting muscle reﬂects

the presence of denervated muscle ﬁbers. This ﬁgure illustrates the extent of spontaneous

EMG activity observed in various muscles of an HCSMA homozygote aged about 160 days.

EMG activity was recorded with a standard, bipolar, concentric needle electrode with the

animal maintained under general anesthesia. Spontaneous activity was graded using a standard

clinical rating scale of 0 (no spontaneous activity) to 4 (maximum activity). 57 Denervation

appears ﬁrst in tail muscles at about 10 weeks of age and spreads in rostral and distal directions

so that distal hindlimb and forelimb muscles do not exhibit signs of denervation until late in

the course of the disease. In the case of ankle extensors, experiments have shown that

signiﬁcant motor unit dysfunction exists by 160 days despite the absence of spontaneous

EMG activity.

(HCSMA). HCSMA is an inherited disorder of motor neurons that shares features

with human motor neuron disease. 1,14 Genetic studies have shown that HCSMA is

an autosomal dominant disorder. 15 Although the defective gene has not yet been

identiﬁed, a recent study 16 has demonstrated that HCSMA is not caused by the

mutations in the survival motor neuron (SMN) gene that are responsible for the

human spinal muscular atrophies. 17

Consistent with its autosomal dominant inheritance, disease in HCSMA mani-

fests as two phenotypes. An accelerated phenotype (presumed homozygous) begins

showing weakness about 6 to 8 weeks postnatal with rapid and progressive deteri-

oration that culminates in full tetraparesis by about 6 to 7 months of age. HCSMA

homozygotes do not survive to reach sexual maturity. Homozygotes exhibit a ste-

reotyped spatiotemporal pattern of muscle denervation which is ﬁrst observed in

caudal tail muscles. Subsequently, denervation spreads in a rostral and distal direc-

tion so that distal hindlimb/forelimb muscles are the last to become involved. An

illustration of the spatial distribution and extent of spontaneous electromygraphic

(EMG) activity caused by muscle denervation is shown in Fig. 10.1 for an HCSMA

homozygote aged 160 days. A more chronic phenotype (presumed heterozygote) begins

FIGURE 10.2 Motor neuron cell death in an HCSMA heterozygote. Photomicrograph shows

a cross-section of lumbar spinal cord from an HCSMA heterozygote aged 5 years. The solid

line indicates the border between gray and white matter. Note the absence of large motor

neuron cell bodies in the ventral aspect of the spinal gray.

showing weakness at about 8 to 12 months and can survive as long as 7 years. The

development of muscle denervation in heterozygotes occurs over a much more pro-

tracted time course but exhibits essentially the same spatial pattern as homozygotes.

Histological studies of the brain and spinal cord indicate that pathological

involvement in HCSMA is limited to motor neurons. 18 Whether motor neurons are

exclusively involved is not known, however, because of uncertainty about whether

full disease expression is achieved during the time interval over which these animals

can be humanely maintained. More extensive involvement is found, for example, in

ALS patients who are maintained for extended periods by artiﬁcial ventilation. 19

This illustrates an important point: Limited observations of progressive disorders,

perhaps even over the natural disease course, may lead to erroneous conclusions

concerning the selectivity of involved neuronal populations.

The pathological features found among motor neurons in HCSMA are similar

to those found in other versions of motor neuron disease. These features include

abnormal neuroﬁlament accumulations in proximal motor axons, motor neuron

chromatolysis, and neuronophagia, all considered evidence of diseased motor neu-

rons. 18 One common feature of motor neuron disease that is not observed in HCSMA

homozygotes during the time they can be studied (up to about 6 months) is extensive

motor neuron cell death. 18 Since weakness progresses to the point of tetraparesis

during this interval, it is apparent that mechanisms other than motor neuron cell

death must mediate the progressive weakness in HCSMA homozygotes. As illus-

trated in Fig. 10.2 , however, loss of motor neurons is observed in heterozygotes thus

demonstrating that motor neuron cell death is a feature of HCSMA. The relative

absence of motor neuron cell death in homozygotes may be related to the accelerated

progression rate of weakness in these animals (particularly among neck and proximal

limb extensor muscle groups, Fig. 10.1 ) which renders them moribund before cell

death has an opportunity to appear. The much longer time period over which het-

erozygotes develop symptoms and weakness (years vs. about 2 to 3 months for

homozygotes) presumably enables a more complete expression of the disorder.

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