**8. Conclusion**

Motor neurone disease (MND) is a group of fatal, progressive neurodegenerative disorders, with an overall median survival is approximately 4.0 years from the onset of symptoms. By the time most patients with MND are aware of clinical weakness and seek review by their primary physician or neurologist, a significant proportion of motor units have already been lost. Early detection of motor neurone loss in clinically apparently unaffected muscles is therefore important to establish an early diagnosis of the condition.

Motor unit number estimates in the group of asymptomatic SOD1 mutation carriers were compared to age and sex matched family controls without the SOD1 mutation, and sporadic (non-SOD1) MND patients. There was no detectable difference in the number of thenar motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (thenar p>0.46), or population controls (thenar p>0.70).. In addition, there was no detectable difference in the number of EBD motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50). Symptomatic sporadic MND subjects showed a definite loss of motor units with fewer motor units compared to all other groups (p<0.001). There was no overlap between MUNE results in symptomatic and asymptomatic subjects.

These results indicate that the group of asymptomatic carriers of the SOD1 mutation have no significant difference in the number of motor neurones, when compared to age and sex matched controls. All carriers had a full complement of motor neurones during the asymptomatic phase, indicating that mutation carriers have normal survival of motor neurones and that symptomatic MND is not the end result of a slow attrition of motor neurones. This implies that gradual pre-symptomatic loss of motor neurones does not occur in asymptomatic SOD1 mutation carriers. This supports the observation that sudden, catastrophic loss of motor neurones occurs immediately prior to the onset of symptoms and the development of the disease, rather than a gradual attrition of motor neurones over time. These results suggest that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms. This observation is an important contribution to the current understanding of the pathogenesis of MND. (Aggarwal & Nicholson, 2001).

The statistical MUNE technique was used for the study. This technique has been greatly modified since its original description, but numerous studies have shown that MUNE can

as to when weakness developed. Needle EMG showed changes of active and chronic denervation limited to the right quadriceps muscle, which was not considered diagnostic for

Over the next 6 months, there was a reduction of the left EDB MUNE as well from 114 to 36 (68% reduction), but with no detectable weakness. In July 2002, 8 months later, he was reviewed by his neurologist and was found to have only slightly reduced left ankle power with MRC grade 4/5 and weakness of knee flexion bilaterally. He progressed rapidly after that and by October 2002 had bilateral lower limb weakness to a point were he was unable to stand without assistance and became wheelchair bound. He commenced Riluzole, in May 2003, but over the next year there was progression of upper limb weakness. Currently, his forced lung capacity is around 30% and he is using BiPAP ventilation at night and receives

Motor neurone disease (MND) is a group of fatal, progressive neurodegenerative disorders, with an overall median survival is approximately 4.0 years from the onset of symptoms. By the time most patients with MND are aware of clinical weakness and seek review by their primary physician or neurologist, a significant proportion of motor units have already been lost. Early detection of motor neurone loss in clinically apparently unaffected muscles is

Motor unit number estimates in the group of asymptomatic SOD1 mutation carriers were compared to age and sex matched family controls without the SOD1 mutation, and sporadic (non-SOD1) MND patients. There was no detectable difference in the number of thenar motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (thenar p>0.46), or population controls (thenar p>0.70).. In addition, there was no detectable difference in the number of EBD motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50). Symptomatic sporadic MND subjects showed a definite loss of motor units with fewer motor units compared to all other groups (p<0.001). There was no overlap between MUNE results in symptomatic and

These results indicate that the group of asymptomatic carriers of the SOD1 mutation have no significant difference in the number of motor neurones, when compared to age and sex matched controls. All carriers had a full complement of motor neurones during the asymptomatic phase, indicating that mutation carriers have normal survival of motor neurones and that symptomatic MND is not the end result of a slow attrition of motor neurones. This implies that gradual pre-symptomatic loss of motor neurones does not occur in asymptomatic SOD1 mutation carriers. This supports the observation that sudden, catastrophic loss of motor neurones occurs immediately prior to the onset of symptoms and the development of the disease, rather than a gradual attrition of motor neurones over time. These results suggest that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms. This observation is an important contribution to the current

The statistical MUNE technique was used for the study. This technique has been greatly modified since its original description, but numerous studies have shown that MUNE can

understanding of the pathogenesis of MND. (Aggarwal & Nicholson, 2001).

therefore important to establish an early diagnosis of the condition.

ALS.

PEG feeding.

**8. Conclusion**

asymptomatic subjects.

change systematically in ALS patients when used by experienced technicians, even though evaluator bias needs to be taken into account. Shefner demonstrated that the statistical MUNE was unreliable in the presence of clinical weakness due to motor unit instability. The difference is that our study was performed on asymptomatic patients, without clinical weakness.

It also showed that MUNE may be used as a reliable method of pre-symptomatic detection of motor unit loss in SOD1 mutation carriers. Following 69 SOD1 family members and population controls over a 1-year period, with thenar and EDB MUNE tests repeated every 3 to 6 months, assessed the test-retest reproducibility of the technique. The mean difference between thenar MUNE results on separate occasions in asymptomatic subjects was +/- 3.6%, with a range of 0-11.7%, and +/- 4.6%, with a range of 0-15.7% in EDB MUNE. These results indicate that the reproducibility of this technique and the results achieved was high, so that individual results could be used as a baseline for serial MUNE studies. (Aggarwal, 2009).

During the course of the study, however, a significant fall in motor unit number was detected in 5 of the SOD1 mutation carriers, several months before the onset of weakness and the diagnosis of motor neurone disease (MND) being made. There was no detectable loss of motor units in the other 15 SOD1 mutation carriers or in the group of SOD1 mutation negative relatives. From the study, a threshold MUNE of less than 100 was considered to imply that symptoms were imminent.

In individual cases, there was a reduction of 68% 8 months prior, 51% 4 months prior, 46% 6 months prior, 35% 10 months prior and 28% 6 months prior to the onset of weakness. Further motor unit loss occurred as weakness progressed and the diagnosis of MND being made.

Case 1 was a 48-year-old lady from a family with a strong history of familial MND, with a point mutation in the SOD1 gene at val148gly. At the time of recruitment in October 1998, she was asymptomatic. Her MUNE results remained stable over the first 2½ years, after which her left EDB MUNE dropped by 51%, and she only had detectable weakness of her left foot 4 months later with wasting and weakness of the anterior compartment muscles of her left leg of MRC grade 2-3/5. Over the next 6 months, her right EDB MUNE dropped by 56%, but she only developed right foot weakness 3 months later in June2001.

Her 43-year-old sister also showed a reduction in MUNE prior to the onset of symptoms. About 3 years into the study, there was a reduction in her right thenar MUNE to 96 (20%) and her left thenar MUNE to 89 (19%), with no detectable weakness. Her right EDB MUNE also dropped by 17%, but she only had detectable weakness 10 months later of MRC grade 4+/5 in right dorsiflexors, at which time her right EDB MUNE had dropped by a total of 35%. In view of her strong family history, a presumed diagnosis of MND was made and she was commenced on Riluzole in February 2002. Over the last 2 years, her EDB MUNE have not shown any decline. Her weakness has not progressed significantly, as on her last review in February 2004, she still had MRC grade 4/5 power of her right dorsiflexors and no clinically apparent weakness in her left dorsiflexors or upper limbs. It is possible that since "treatment" was commenced prior to the loss of a significant number of motor neurones, this may have slowed down the progression of the disease in this individual case. (Aggarwal & Nicholson, 2002).

Her mother also had a detectable reduction right thenar MUNE of 23% and left thenar MUNE of 25%, with no clinically apparent weakness. Her right EDB MUNE also dropped by 28%, with no detectable weakness. She subsequently developed bulbar symptoms 6 months and died of respiratory failure.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

vital capacity and Medical Research Council manual muscle testing.

Riluzole is a disease altering agent nor does it have any restorative reports.

In one of the cases in the study, Riluzole was commenced once she developed mild weakness. At the time, there was a slight reduction in MUNE, but conventional needle EMG examination did not fulfil the criteria to make the diagnosis of MND. In view of her strong family history and positive genetic testing, a presumed diagnosis of MND was made. Since commencing Riluzole there has been no significant reduction in her EDB MUNE over the last 2 years, and her weakness of right dorsiflexors has only progressed marginally from MRC grade 4+/5 to 4/5 power. It is possible that since "treatment" was commenced prior to the loss of a significant number of motor neurones, this may have slowed down the progression of the disease in this individual case. Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms

voluntary isometric contraction (MVIC).

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 657

Maximum isometric grip strength using the Jamar hydraulic dynamometer also does not correlate with the number of functional motor neurones in thenar group of muscles as measured using the statistical method of MUNE, indicating that MUNE is a more sensitive test than MVIC for monitoring disease progression in MND. It has also been shown that MUNE is able to identify deterioration in functional motor units before handgrip maximal

This confirms McComas' observation that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units occurs. This suggests that handgrip MVIC is not as sensitive as thenar MUNE for monitoring disease progression, as it is unable to detect early motor neurone loss due to the presence of compensatory mechanisms. The surviving motor neurones enlarge their territories, through collateral sprouting (reinnervation) until late in the disease, when collateral reinnervation is no longer able to provide full functional compensation. Thenar MUNE however does examine all of the motor units that are involved in handgrip MVIC, as forearm flexors and ulnar-innervated muscles are involved in the generation of handgrip MVIC. It also confirms Felice's study which showed that in patients with MND, changes in thenar MUNE was the most sensitive outcome measure for following disease progression, when compared to other quantitative tests, such as CMAP, isometric grip strength, forced

As motor neurone loss once it occurs is rapid and precipitous, any potential treatment will need to be given early to SOD1 mutation carriers. Once the disease progresses, resulting in functional impairment and disability, restorative treatments to replace lost motor neurones becomes less feasible. To date there have been a number of drugs which have undergone clinical trials in MND, for which there is no evidence of benefit. These include creatinine, high dose vitamin E, Gabapentin and nerve growth factors such as brain derived neurotrophic factor and insulin-like growth factor-1. If effective treatment for MND were to be developed to arrest the process of degeneration, therapies aimed at preserving functional motor neurones would be more feasible. This requires the ability to be able to identify individuals at risk of developing the disease, which currently are SOD1 mutation carriers. Currently, the only effective approved treatment for MND is Riluzole, which has a neuroprotective role, possibly due to pre-synaptic inhibition of glutamate release. Riluzole is an anti-glutamate agent that has been approved for the treatment of patients with amyotrophic lateral sclerosis in most countries. There have been a least three large randomised trials involving hundreds of patients that have been unable to show that

Case 4 was a 48-year-old lady with a strong family history of MND and a point mutation in SOD1 gene at glu100gly. At the time of recruitment, she was asymptomatic and her MUNE results remained stable over the first 3 years of the study. It was then noted that her reduction of her left EDB MUNE of 46% reduction, but with no detectable weakness. She subsequently developed left lower leg weakness of MRC grade 3/5 about 6months later.

Finally, her 2nd cousin, once removed, is a 44-year-old man who also had a point mutation in SOD1 gene at glu100gly. His MUNE results also remained stable over the first 3 years of the study. The right EDB MUNE dropped from 115 down to being not recordable over a 12 month period. At that time, he had occasional fasciculations in the right quadriceps region and MRC grade 0/5 weakness of right dorsiflexion. He was unsure as to when the weakness had developed. Over time, his left EDB MUNE has reduced by 68%, but his left ankle power remained normal until 8 months later, when it reduced slightly to MRC grade 4/5.

This study also shows that there can be substantial loss in MUNE and still have an essentially normal EMG with minimal signs of acute denervation or motor unit potential remodelling, as one would expected that at a minimum, the muscles with transiently reduced MUNE numbers should have reduced recruitment during EMG studies.

Four of the five SOD 1 mutation carriers who had a pre-symptomatic loss of motor neurones had needle EMG studies performed by an independent neurologist, which showed neurogenic changes but not sufficient to fulfil the criteria to make the diagnosis of MND. All had a reduction in MUNE at the time of the EMG study of which the independent neurologist was not aware. This implies that MUNE may be a more reliable and sensitive method for diagnosing MND than needle EMG. MUNE can be used as a non-invasive method of predicting impending decline in motor neurones and estimating the rate of neuronal death in asymptomatic subjects. This indicates that loss of motor neurones is detectable in the pre-symptomatic phase and this loss was detectable before significant needle EMG changes of pathology occur. McComas showed that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional compensation, and this is the probable explanation of this finding.

As MUNE is a measure of the primary pathologic process of motor neurone loss and can identify that the number of motor units are reduced, even in the presence of a nondiagnostic needle EMG. Needle electromyography may reveal evidence of chronic reinnervation, but provides little direct evidence to the extent of motor neurone and axonal loss.

This lack of corroboration with needle EMG in the pre-symptomatic stage requires a paradigm shift in the traditional concept that needle EMG is the "gold" standard for the diagnosis of ALS. We are aware that traditional neurologists and neurophysiologists will find this difficult to accept, as it would be expected that at a minimum, the muscles with transiently reduced MUNE numbers should have reduced recruitment during EMG studies. It is hard to understand physiologically how there can be substantial loss in MUNE and still have normal EMG with no signs of acute denervation or motor unit potential remodelling. These cases clearly indicate that loss of motor neurones is detectable in the pre-symptomatic phase and this loss was detectable before significant needle EMG changes of pathology occur. Even though some may argue that a reduction in MUNE cannot be used to support or diagnosis FALS, once changes occur on conventional EMG studies, the window of opportunity to influence the progression of this condition has been missed.

Case 4 was a 48-year-old lady with a strong family history of MND and a point mutation in SOD1 gene at glu100gly. At the time of recruitment, she was asymptomatic and her MUNE results remained stable over the first 3 years of the study. It was then noted that her reduction of her left EDB MUNE of 46% reduction, but with no detectable weakness. She subsequently developed left lower leg weakness of MRC grade 3/5 about 6months later. Finally, her 2nd cousin, once removed, is a 44-year-old man who also had a point mutation in SOD1 gene at glu100gly. His MUNE results also remained stable over the first 3 years of the study. The right EDB MUNE dropped from 115 down to being not recordable over a 12 month period. At that time, he had occasional fasciculations in the right quadriceps region and MRC grade 0/5 weakness of right dorsiflexion. He was unsure as to when the weakness had developed. Over time, his left EDB MUNE has reduced by 68%, but his left ankle power

remained normal until 8 months later, when it reduced slightly to MRC grade 4/5.

reduced MUNE numbers should have reduced recruitment during EMG studies.

compensation, and this is the probable explanation of this finding.

opportunity to influence the progression of this condition has been missed.

loss.

This study also shows that there can be substantial loss in MUNE and still have an essentially normal EMG with minimal signs of acute denervation or motor unit potential remodelling, as one would expected that at a minimum, the muscles with transiently

Four of the five SOD 1 mutation carriers who had a pre-symptomatic loss of motor neurones had needle EMG studies performed by an independent neurologist, which showed neurogenic changes but not sufficient to fulfil the criteria to make the diagnosis of MND. All had a reduction in MUNE at the time of the EMG study of which the independent neurologist was not aware. This implies that MUNE may be a more reliable and sensitive method for diagnosing MND than needle EMG. MUNE can be used as a non-invasive method of predicting impending decline in motor neurones and estimating the rate of neuronal death in asymptomatic subjects. This indicates that loss of motor neurones is detectable in the pre-symptomatic phase and this loss was detectable before significant needle EMG changes of pathology occur. McComas showed that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units, before collateral reinnervation was unable to provide functional

As MUNE is a measure of the primary pathologic process of motor neurone loss and can identify that the number of motor units are reduced, even in the presence of a nondiagnostic needle EMG. Needle electromyography may reveal evidence of chronic reinnervation, but provides little direct evidence to the extent of motor neurone and axonal

This lack of corroboration with needle EMG in the pre-symptomatic stage requires a paradigm shift in the traditional concept that needle EMG is the "gold" standard for the diagnosis of ALS. We are aware that traditional neurologists and neurophysiologists will find this difficult to accept, as it would be expected that at a minimum, the muscles with transiently reduced MUNE numbers should have reduced recruitment during EMG studies. It is hard to understand physiologically how there can be substantial loss in MUNE and still have normal EMG with no signs of acute denervation or motor unit potential remodelling. These cases clearly indicate that loss of motor neurones is detectable in the pre-symptomatic phase and this loss was detectable before significant needle EMG changes of pathology occur. Even though some may argue that a reduction in MUNE cannot be used to support or diagnosis FALS, once changes occur on conventional EMG studies, the window of Maximum isometric grip strength using the Jamar hydraulic dynamometer also does not correlate with the number of functional motor neurones in thenar group of muscles as measured using the statistical method of MUNE, indicating that MUNE is a more sensitive test than MVIC for monitoring disease progression in MND. It has also been shown that MUNE is able to identify deterioration in functional motor units before handgrip maximal voluntary isometric contraction (MVIC).

This confirms McComas' observation that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 70-80% of motor units occurs. This suggests that handgrip MVIC is not as sensitive as thenar MUNE for monitoring disease progression, as it is unable to detect early motor neurone loss due to the presence of compensatory mechanisms. The surviving motor neurones enlarge their territories, through collateral sprouting (reinnervation) until late in the disease, when collateral reinnervation is no longer able to provide full functional compensation. Thenar MUNE however does examine all of the motor units that are involved in handgrip MVIC, as forearm flexors and ulnar-innervated muscles are involved in the generation of handgrip MVIC. It also confirms Felice's study which showed that in patients with MND, changes in thenar MUNE was the most sensitive outcome measure for following disease progression, when compared to other quantitative tests, such as CMAP, isometric grip strength, forced vital capacity and Medical Research Council manual muscle testing.

As motor neurone loss once it occurs is rapid and precipitous, any potential treatment will need to be given early to SOD1 mutation carriers. Once the disease progresses, resulting in functional impairment and disability, restorative treatments to replace lost motor neurones becomes less feasible. To date there have been a number of drugs which have undergone clinical trials in MND, for which there is no evidence of benefit. These include creatinine, high dose vitamin E, Gabapentin and nerve growth factors such as brain derived neurotrophic factor and insulin-like growth factor-1. If effective treatment for MND were to be developed to arrest the process of degeneration, therapies aimed at preserving functional motor neurones would be more feasible. This requires the ability to be able to identify individuals at risk of developing the disease, which currently are SOD1 mutation carriers.

Currently, the only effective approved treatment for MND is Riluzole, which has a neuroprotective role, possibly due to pre-synaptic inhibition of glutamate release. Riluzole is an anti-glutamate agent that has been approved for the treatment of patients with amyotrophic lateral sclerosis in most countries. There have been a least three large randomised trials involving hundreds of patients that have been unable to show that Riluzole is a disease altering agent nor does it have any restorative reports.

In one of the cases in the study, Riluzole was commenced once she developed mild weakness. At the time, there was a slight reduction in MUNE, but conventional needle EMG examination did not fulfil the criteria to make the diagnosis of MND. In view of her strong family history and positive genetic testing, a presumed diagnosis of MND was made. Since commencing Riluzole there has been no significant reduction in her EDB MUNE over the last 2 years, and her weakness of right dorsiflexors has only progressed marginally from MRC grade 4+/5 to 4/5 power. It is possible that since "treatment" was commenced prior to the loss of a significant number of motor neurones, this may have slowed down the progression of the disease in this individual case. Early in the course of ALS, the rate of cell death is low as the amount of neuronal damage caused by the mutation is small. As the amount of intracellular damage increases, a critical threshold is reached, which overwhelms

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

mutation carriers.

after early clinical diagnosis.

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 659

by the mutation is small. The delay in clinical onset was thought to reflect the gradual accumulation of damage within the neurones, as a result of the mutation, which ultimately overwhelms cellular homeostasis leading to cell death. The living mutant neurons function very well for years or decades but the probability that an individual neurone undergoes apoptosis increases as damage accumulates within it. A mutant neurone in an older patient will have accumulated a greater amount of damage and will therefore be more likely to die than in a younger patient. Consequently, early in the course of disease, the chance of a cell containing a sufficient amount of damage to initiate apoptosis is small, and the rate of cell loss is correspondingly low. The mutant neurones appear to function normally for decades, with weakness only occurring once apoptosis and cell death occurs due to a gradual accumulation of damage within the cell. Therapies aimed at preserving motor neurones may be more feasible than trying to replace lost motor neurones. A number of treatment or preventative strategies arise, such as measures to diminish SOD1 aggregation or interactions to specifically reduced apoptosis in motor neurones. As motor neurone loss at this stage is rapid and precipitous, any potential treatment will need to be given much earlier in SOD1

Determining the mechanism by which mutations in the Cu/Zn superoxide dismutase (SOD1) gene triggers the destruction of motor neurones causing MND remains unknown. At present, the favoured hypothesis is that the mutation causes disease as a result of a toxic gain of function by the mutant SOD1 provoking selective neurotoxicity, probably disrupting the intracellular homeostasis of copper and/or protein aggregation. However, as the amount of intracellular damage increases, the chance that a cell will die also increases. This cumulative damage may be due to oxidative stress, in which an imbalance between the production of reactive oxygen species and cellular antioxidant mechanisms results in chemical modifications of macromolecules, thereby disrupting cellular structure and function. It is possible that the high metabolic activity in motor neurones, combined with the toxic oxidative properties of the mutant SOD1, causes massive mitochondrial vacuolation in motor neurones, resulting in degeneration, earlier than other neurones, triggering the onset of weakness. Prominent cytoplasmic intracellular inclusions in motor neurones and within astrocytes surrounding them developed by the onset of clinical disease and in some cases represented the first pathological sign of disease. These aggregates increased in number as the disease progressed. This indicates that the mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurones and this damage triggers the functional decline of motor neurones and the clinical onset of symptoms. The absence of motor neurone death in the early stages of the disease indicates that the majority of motor neurones could be rescued

Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones. If the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS. At this stage, detecting the onset of motor neurone loss in asymptomatic individuals will identify those who may benefit from early institution of an active management program to improve their quality of life, until more effective treatment modalities become available for this devastating condition This observation is an important contribution to the current understanding of the pathogenesis of MND, as it shows that motor neurone disease does not seem to be the end result of slow attrition of motor neurones. MUNE may be able to be used

cellular homeostasis, resulting in rapid apoptosis and cell death. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

We would argue that previous trials have all be performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the presymptomatic phase of the disease, when the therapeutic benefit might change, as "treatment" is commenced prior to significant motor neurone loss occurring and therefore, the progression of disease can be slowed down. MUNE numbers are believed to reduce because of remodelling of the motor unit and in our study, the compound muscle action potential amplitudes (CMAP) were retained as early in the course of the disease, the rate of cell death is low. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in CMAP.

This longitudinal study showed that it was possible to detect loss of motor neurones in the pre-symptomatic stage of MND in humans. This study provided further evidence that considerable motor neurone loss occurred just before the onset of symptoms or weakness. (Aggarwal, 2009).

This study indicates that SOD1 mutation carriers have normal survival of motor neurones, with as carriers had a full complement of motor neurones during the asymptomatic phase. Significant pre-symptomatic loss of motor neurones did not occur in asymptomatic SOD1 mutation carriers. Sudden and widespread motor neurone death occurs at the time development of the symptomatic symptoms, rather than life-long motor neurone loss. Sudden, catastrophic and multifocal loss of motor neurons occurs immediately prior to the onset of symptoms and the development of MND. This suggests that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms, rather than life-long motor neurone loss. Also, if the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS and develop treatments for sporadic MND. The mutant SOD1 protein itself cannot be the trigger, as it is constantly expressed. There may however be a gradual accumulation of a toxic product, possibly SOD1, which has changed into a new toxic conformation or aggregate, resulting in neuronal damage. The possibility of an individual neuron undergoing apoptosis increases as damage accumulates . This cumulative damage may be due to oxidative stress, resulting in disruption of the cellular structure and function.

Neurofilament heavy polypeptide (NF-H) is an abundant stable cytoplasmic protein located in neuronal cells in large axons and may be used as a cell type marker. Abnormal accumulation of NF-H in motor neurones is associated with ALS, but it is unclear to what extent these contribute to human disease. Analysis of blood serum markers looking for increased levels of NF-H was not performed in this study, but would be interesting to be done in the future to the compare levels of NF-H in the carriers.

The results of this study indicate that the risk of cell death probably remains constant throughout life of the neurone and that cell death occurs randomly in time and is independent of that of any other neurone. This suggests a "one-hit" biochemical phenomenon in which the mutation imposes an abnormal mutant steady state on the neurone and a single catastrophic event randomly initiates cell death and apoptosis. Early in the course of MND, the rate of cell death is low as the amount of neuronal damage caused

cellular homeostasis, resulting in rapid apoptosis and cell death. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease, without resulting in a change in

We would argue that previous trials have all be performed in the symptomatic phase of the disease when 70-80% of motor units have already been lost, rather than in the presymptomatic phase of the disease, when the therapeutic benefit might change, as "treatment" is commenced prior to significant motor neurone loss occurring and therefore, the progression of disease can be slowed down. MUNE numbers are believed to reduce because of remodelling of the motor unit and in our study, the compound muscle action potential amplitudes (CMAP) were retained as early in the course of the disease, the rate of cell death is low. The increase in MUNE numbers may be either due to reinnervation of the damaged muscle or repair of poorly functioning synapses, at the early stage of the disease,

This longitudinal study showed that it was possible to detect loss of motor neurones in the pre-symptomatic stage of MND in humans. This study provided further evidence that considerable motor neurone loss occurred just before the onset of symptoms or weakness.

This study indicates that SOD1 mutation carriers have normal survival of motor neurones, with as carriers had a full complement of motor neurones during the asymptomatic phase. Significant pre-symptomatic loss of motor neurones did not occur in asymptomatic SOD1 mutation carriers. Sudden and widespread motor neurone death occurs at the time development of the symptomatic symptoms, rather than life-long motor neurone loss. Sudden, catastrophic and multifocal loss of motor neurons occurs immediately prior to the onset of symptoms and the development of MND. This suggests that there may be a biological trigger initiating rapid cell loss, just prior to the onset of symptoms, rather than life-long motor neurone loss. Also, if the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS and develop treatments for sporadic MND. The mutant SOD1 protein itself cannot be the trigger, as it is constantly expressed. There may however be a gradual accumulation of a toxic product, possibly SOD1, which has changed into a new toxic conformation or aggregate, resulting in neuronal damage. The possibility of an individual neuron undergoing apoptosis increases as damage accumulates . This cumulative damage may be due to oxidative stress, resulting

Neurofilament heavy polypeptide (NF-H) is an abundant stable cytoplasmic protein located in neuronal cells in large axons and may be used as a cell type marker. Abnormal accumulation of NF-H in motor neurones is associated with ALS, but it is unclear to what extent these contribute to human disease. Analysis of blood serum markers looking for increased levels of NF-H was not performed in this study, but would be interesting to be

The results of this study indicate that the risk of cell death probably remains constant throughout life of the neurone and that cell death occurs randomly in time and is independent of that of any other neurone. This suggests a "one-hit" biochemical phenomenon in which the mutation imposes an abnormal mutant steady state on the neurone and a single catastrophic event randomly initiates cell death and apoptosis. Early in the course of MND, the rate of cell death is low as the amount of neuronal damage caused

CMAP.

(Aggarwal, 2009).

without resulting in a change in CMAP.

in disruption of the cellular structure and function.

done in the future to the compare levels of NF-H in the carriers.

by the mutation is small. The delay in clinical onset was thought to reflect the gradual accumulation of damage within the neurones, as a result of the mutation, which ultimately overwhelms cellular homeostasis leading to cell death. The living mutant neurons function very well for years or decades but the probability that an individual neurone undergoes apoptosis increases as damage accumulates within it. A mutant neurone in an older patient will have accumulated a greater amount of damage and will therefore be more likely to die than in a younger patient. Consequently, early in the course of disease, the chance of a cell containing a sufficient amount of damage to initiate apoptosis is small, and the rate of cell loss is correspondingly low. The mutant neurones appear to function normally for decades, with weakness only occurring once apoptosis and cell death occurs due to a gradual accumulation of damage within the cell. Therapies aimed at preserving motor neurones may be more feasible than trying to replace lost motor neurones. A number of treatment or preventative strategies arise, such as measures to diminish SOD1 aggregation or interactions to specifically reduced apoptosis in motor neurones. As motor neurone loss at this stage is rapid and precipitous, any potential treatment will need to be given much earlier in SOD1 mutation carriers.

Determining the mechanism by which mutations in the Cu/Zn superoxide dismutase (SOD1) gene triggers the destruction of motor neurones causing MND remains unknown. At present, the favoured hypothesis is that the mutation causes disease as a result of a toxic gain of function by the mutant SOD1 provoking selective neurotoxicity, probably disrupting the intracellular homeostasis of copper and/or protein aggregation. However, as the amount of intracellular damage increases, the chance that a cell will die also increases. This cumulative damage may be due to oxidative stress, in which an imbalance between the production of reactive oxygen species and cellular antioxidant mechanisms results in chemical modifications of macromolecules, thereby disrupting cellular structure and function. It is possible that the high metabolic activity in motor neurones, combined with the toxic oxidative properties of the mutant SOD1, causes massive mitochondrial vacuolation in motor neurones, resulting in degeneration, earlier than other neurones, triggering the onset of weakness. Prominent cytoplasmic intracellular inclusions in motor neurones and within astrocytes surrounding them developed by the onset of clinical disease and in some cases represented the first pathological sign of disease. These aggregates increased in number as the disease progressed. This indicates that the mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurones and this damage triggers the functional decline of motor neurones and the clinical onset of symptoms. The absence of motor neurone death in the early stages of the disease indicates that the majority of motor neurones could be rescued after early clinical diagnosis.

Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones. If the trigger initiating motor neurone loss can be identified, it may be possible to prevent motor neurone loss in familial ALS. At this stage, detecting the onset of motor neurone loss in asymptomatic individuals will identify those who may benefit from early institution of an active management program to improve their quality of life, until more effective treatment modalities become available for this devastating condition This observation is an important contribution to the current understanding of the pathogenesis of MND, as it shows that motor neurone disease does not seem to be the end result of slow attrition of motor neurones. MUNE may be able to be used

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

*Neurol Neurosurg Psychiatry*. 36: 174-182.

degenerations. *Nature.* 406: 195-199.

death in ALS. *Neuron.* 24: 515-520.

633-640.

41: 210-221.

S233-241.

18:741-752.

20: 179-185.

2000; 288: 283-284.

12(6): 585-594.

*Genet.* 32: 841-847.

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 661

Bruijn, L.I., Becher, M.W., Lee, M.K., Anderson, K.L., Jenkins, N.A., Copeland, N.G., Sisodia,

progressive disease with SOD1-containing inclusions. *Neuron*. 18: 327-338. Campbell, M.J., McComas, A.J. & Petito, F. (1973). Physiological changes in ageing muscles. *J*

Chance, P.F., Rabin, B.A., Ryan, S.G., Ding, Y., Scavina, M., Crain, B., Griffith, J.W.,

Cheah, B.C., Vucic, S., Krishnan, A.V., Kiernan, M.C. (2010). Riluzole, neuroprotection and

Clarke, G., Collins, R.A., Leavitt, B.R., Andrews, D.F., Hayden, M.R., Lumsden, C.J. &

Clarke, G., Lumsden, C.J. & McInnes, R.R. (2001). Inherited neurodegenerative disease: the one hit model of neurodegeneration. *Human Molecular Genetics.* 10: 2269-2275. Cleveland, D.W. (1999). From Charcot to SOD1: Mechanisms of selective motor neuron

Cudkowicz, M.E., McKenna-Yasek, D., Sapp, P.E., Chin, W., Geller, B., Hayden, D.L.,

Daube, J.R. (1995). Estimating the number of motor units in a muscle. *J Clin Neurophysiol*.

de Belleroche, J., Orrell, R., King, A. J. (1995). Medical Genetics. Familial amyotrophic lateral

Doble, A. (1996). The pharmacology and mechanism of action of riluzole. *Neurology*. 47:

Eisen, A. (1995). Amyotrophic lateral sclerosis is a multifactorial disease. *Muscle Nerve*.

Felice, K.J. (1997). A longitudinal study comparing thenar motor unit number estimation to

Figlewicz, D.A., Krizus, A., Martinoli, M.G., Meininger, V., Dib, M., Rouleau, G.A., Julein,

development of amyotrophic lateral sclerosis. *Hum Mol Genet* 3: 1757-1761. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D.,

Gurney, M.E., Tomasselli, A.G., Heinrikson, R.L. (1994). Stay the Executioner's hand. *Science*

Hamilton, A., Balnave, R. & Adam, R. Grip strength reliability. *J Hand Therapy.* 7(3): 163-170.

superoxide dismutase mutation. *Science*. 264: 1772-1775.

amyotrophic lateral sclerosis. *Curr Med Chem* 17(18): 1942-49.

S.S., Rothstein, J.D., Borchelt, D.R., Price, D.L.& Cleveland, D.W. (1997). ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly

Cornblath, D.R. (1998). Linkage of the gene for an autosomal dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34. *Am J Hum Genet.* 62:

McInnes, R,R. (2000). A one hit model of cell death in inherited neuronal

Schoenfeld, D.A., Hosler, B.A., Horvitz, H.R., Brown, R.H. Jr. (1997). Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. *Ann Neurol.*

sclerosis/motor neurone disease (FALS): a review of current developments. *J Med*

other quantitative tests in patients with amyotrophic lateral sclerosis. *Muscle Nerve*.

J.P. (1994). Variants of the heavy neurofilament subunit are associated with the

Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H.X., Chen, W., Zhai, P., Sufit, R.L. & Siddique, T. (1994). Motor neuro degeneration in mice that express a human Cu/Zn

as a method of pre-symptomatic testing of individuals who on genetic testing are SOD1 mutation carriers. Regular follow-up of SOD1 carriers with MUNE may lead to early diagnosis, creating an opportunity for future novel approaches and therapies aimed at preserving motor neurones rather than replacing lost motor neurones.