**6. Methods**

634 Amyotrophic Lateral Sclerosis

Mapping of the mutation sites predicted that these mutations destabilise the protein structure, leading to a less active enzyme i.e. "loss of function". This is however not supported by the fact that transgenic mice over expressing SOD1 gene developed disease similar to MND in humans, while those over-expressing normal SOD1 remained unaffected. This suggests that the mutant mice develop the disease independent of the level of SOD1 activity and suggests that the mutant protein itself is selectively toxic to motor neurones and that there is a "gain of toxic function" rather than a "loss of function". (Gurney et al., 1993). Also, although most mutations in SOD1 gene cause decrease in steady state of cytosolic SOD1 activity, Gly37Arg and Asp90Ala, have no significant decrease in SOD1 activity.

As most SOD1 mutations destabilise SOD1 protein (except Asp90Ala), it is possible that the mutant protein, with altered conformation may become unstable and precipitate to form aggregates or inclusions in motor neurons. These aggregates may then disturb normal cell function and lead to cell death. They are easily formed when SOD1 protein stability is decreased because this protein exists in large amounts accounting for 0.5-1% of total cytosolic protein in neurons. Alternations in the length of the coding sequence, folding, solubility or degradation results in the formation of aggregates. (Yim et al., 1990). Structural changes of mutant SOD1 may distort the rim of the electrostatic guidance channel and allow the catalytic site to become exposed and shallow. Molecules that are normally excluded may gain access to the catalytic reactive site. This results in less buffering of copper and zinc,

The nitric oxide (NO) produced by nitric oxide synthase (NOS) reacts spontaneously with O2- to generate peroxynitrite (ONOO-), which nitrosylates proteins leading to damage. Excess NO may also cause an increase in O2- production by inhibition of mitochondrial electron flow, resulting in further generation of peroxynitrite. This facilitates nitrosylation of tyrosine residues of critical cytosolic proteins thus injuring cells. This reaction is copper dependent. The source of free copper may be mutant SOD1, which cannot accept the ion from the copper chaperone (CCS) protein. Mutant SOD1 possibly exhibit metal mediated cytotoxicities by disrupting the intracellular homeostasis of Cu and Zn, which are potential

The target proteins for nitrosylation include the neurofilament (NF) subunits, which may result in abnormal NF accumulation and subsequent disruption of the NF network and axonal transport, as there is a high neurofilament content in motor neurones. It has also been demonstrated that transgenes encoding mutant NF subunits can directly cause selective degeneration and death of motor neuones. (Cleveland, 1999). Conformational changes have been described in the mutations, Ala4Val, Gly37Arg and His6Arg that may affect the rim of

Glutamate is released from the presynaptic terminal activates the glutamate receptor on the postsynaptic cell membrane. It is then cleared from the synaptic cleft by specific glutamate transporters such as EAAT2. (Trotti et al., 1999). Astrocyte (glial cell) dysfunction may result in selective loss of EAAT2, interfering with the normal clearance of glutamate and allowing it to accumulate in the cell membrane and continue to activate the receptor. (Bruijin et al., 1997). Once activated, the glutamate receptor causes a calcium influx and a cascade of toxicity. The neurone does not have the capacity to buffer this efficiently due to a deficiency in calcium binding proteins (CBP's). This results in disturbances in mitochondrial

the electrostatic guidance channel coded by exon 3.(Sjalander et al., 1995).

metabolism and as a consequence, motor neurone cell death. (Beal, 1996).

which then become neurotoxic. (Radunovic & Leigh, 1996).

neurotoxins. (Gurney & Tomasselli, 2000).

(Shaw et al., 1998).

The Department of Molecular Medicine at Concord hospital had a large database of family members with a known family history of MND, who had blood samples collected for DNA, as part of a previous linkage study. From this database, family members were contacted by telephone by the department's genetic counsellor and informed about the study.

The regional committees for Ethics in Medical Research from Central Sydney Area Health Service, Royal North Shore Hospital and Prince Charles Hospital, approved this study.

All individuals participated without knowledge of their mutation status and on the understanding that this would not be revealed to them. Subjects were also aware that the results obtained from the study would not be available to them and that the information would only be used for research purposes. New consents were obtained from all individuals who participated in the study. The neurologist performing the MUNE studies also had no knowledge of their mutation status. The mutation status was only used in the final analysis of results. Subsequently, they were divided into "SOD1 negative family controls" and "asymptomatic SOD1 mutation carriers".

In addition, studies were also carried out on normal individuals, such as department technicians, spouses of SOD1 family members and individuals from the general population who attended MND support meeting and had an interest in helping to advance research into MND. This group was used as "population controls", to test the validity and reproducibility of the MUNE technique used.

Sporadic MND subjects were also initially studied once the MUNE technique had been validated to demonstrate that the MUNE technique used was able to detect a loss of motor neurones, when present. These were used as "positive controls".

### **6.1 Motor unit number estimation**

Motor unit number estimation (MUNE) estimates the number of functioning lower motor neurones innervating a muscle or a group of muscles and is a measure of the primary pathologic process of motor neurone loss. The concept of motor unit number estimation

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

estimation of the MUNE.

French mathematician (1781-1840).

Hoerth & Slawnych, 2003).

the average size of the SMUP's.

axons) is derived by Poisson statistics.

and that it is the same size each time it is activated.

to obtain the most accurate measurement. (Olney et al., 2000).

(McNeil, 1996).

**6.2 Statistical MUNE method** 

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 637

iii. Finally, there is a phenomenon caused "alternation". This refers to fluctuations in the CMAP amplitude of the same motor unit with similar stimulation intensities. The thresholds of the first few motor axons excited are not sufficiently separate from one another, so that when graded increases in the stimulus intensity occur, the motor axons excited often overlap and add more than one SMUP to the CMAP being recorded. This can result in an underestimation of the mean SMUP size, as it may appear that there are 7 or 8 motor units when there are only 2 or 3 present, which in turn results in an over-

We used the statistical electrophysiological technique of motor unit number estimation (MUNE), (Daube, 1998), was used to estimate the number of motor units in thenar and extensor digitorum brevis muscles. The statistical method estimates the average size of SMUP's and the number of motor units in a group of muscles innervated by the nerve being stimulated, based on the normal variation of the submaximal CMAP evoked with constant stimuli. No attempt is made to identify individual motor unit potentials. The method relies on the known relation between the variance of multiple measures of step functions and the size of the individual steps when the steps have a Poisson distribution. S.D. Poisson was a

Poisson statistics are useful when the distribution arising for events occur randomly in time or when small particles are distributed randomly in space. They have been used to calculate the number of quanta released from a nerve terminal at the neuromuscular junction when the individual quanta are too small to be distinguished, as in myasthenia gravis. ( Lomen-

In pure Poisson statistics, the size of a series of measurements is multiples of the size of a single component. In a Poisson distribution there is a discrete asymmetrical distribution in which responses are found at some levels and others where there are no responses (Figure 4).

A pure Poisson distribution has decreasing numbers at higher values. In Poisson distribution, the variance of these 30 measurements is equal to the size of the individual components making up each measurement. The variance can thus provide an estimate of

The statistical method looks only at variance of the CMAP and does not require identification of individual components. It can be used when the sizes of SMUP's are too small to be isolated. The statistical method assumes that each motor unit has a similar size

Sequences of 30 submaximal stimuli are given. The inherent variability of the threshold of individual axons causes variations in the size of the CMAP. The average change in the submaximal CMAP amplitude caused by alternation (addition and subtraction of motor

The occurrence of alternation with changing units that are activated does not modify the accuracy of the statistical method, because the method is a statistical measurement, a different result is found with each series of 30 stimuli. Therefore, multiple trials are needed

Experimental testing with trials of >300 stimuli has shown that repeated measurement of groups of 30 until the standard deviation of the repeated trials is <10% provides a close

(MUNE) originated in 1967. At the time there was no satisfactory method of assessing the extent of denervation in muscles during life. Analysis of the density of the electromyographic interference pattern during maximal effort was not quantitative, and required the full co-operation of the patient.

The principle of MUNE is that if one can measure the mean single motor unit amplitude (SMUP), it is possible to obtain an estimate of the total number of motor units in the muscle. The results achieved were comparable with estimates of alpha motor fibres obtained by counting axons in specimens of motor nerves. (McComas, 1971).

MUNE has been performed in a number of different ways, each with their advantages and limitations. (Stein & Yang, 1990). The choice of technique depends on the speed and simplicity of the technique, as well as its accuracy and reproducibility. Some methods sample a very small proportion of the number of motor units innervating a muscle (typically 10-20). The coefficient of variation associated with different methods range from 10-45%. (McComas, 1991). If the variability is too large, then the technique cannot be used to follow motor unit loss reliably over time.

The way the average single motor unit potential (SMUP) size is obtained distinguishes the several techniques available. Most employ electrical stimulation of the motor nerve to determine the sizes of the SMUP, but a few use needle EMG.

Each method measures both the average size of the potentials generated by single motor units - single motor unit potentials (SMUP) and the size of the compound muscle action potential (CMAP) obtained with maximal stimulation of a motor nerve.

The motor unit number estimate is calculated by:

 Maximum CMAP amplitude or area MUNE=

 Average single motor unit potential SMUP amplitude or area.

Whereas the methods of measuring the average SMUP differ, they have common assumptions about the measurement of the supramaximal CMAP and the measurement of the average SMUP.

i. Maximal stimulation of any peripheral motor nerve activates all the muscles innervated by that nerve distal to the point of stimulation. Therefore, measurements of the CMAP are the summation of activity from multiple muscles and the MUNE is more accurately an estimate of the number of motor units in a group of muscles rather than in a single muscle.

For example, the median CMAP recorded at abductor pollicis brevis (APB) is more correctly a "thenar MUNE", as it is a summation of the activity of APB, opponens pollicis, flexor pollicis brevis, and to a lesser extent, the lateral lumbricals.

Extensor digitorum brevis (EDB) on the other hand, is a muscle innervated by the deep peroneal nerve. The only source of interfering muscle action potential is from extensor hallucis longus, which can be reduced by correct position of the stimulating electrodes. The muscle belly is flat in profile, eliminating deeper motor units as a cause of small potentials. The recording electrode is placed transversely across the innervation zone, resulting in a simple biphasic negative-positive M wave.

ii. The motor unit potentials used in the calculation of the average SMUP are representative of those generated by the total population of units. All methods, select a subset of the total population of motor units, measure their sizes and calculate an average SMUP for that subgroup.

iii. Finally, there is a phenomenon caused "alternation". This refers to fluctuations in the CMAP amplitude of the same motor unit with similar stimulation intensities. The thresholds of the first few motor axons excited are not sufficiently separate from one another, so that when graded increases in the stimulus intensity occur, the motor axons excited often overlap and add more than one SMUP to the CMAP being recorded. This can result in an underestimation of the mean SMUP size, as it may appear that there are 7 or 8 motor units when there are only 2 or 3 present, which in turn results in an overestimation of the MUNE.
