Possible changes in motor neuron discharge characteristics in presymptomatic amyotrophic lateral sclerosis

Although more than 140 years have passed since amyotrophic lateral sclerosis (ALS) was first described and despite intensive research worldwide, the etiology of the disease is still unknown and there is no effective treatment that will slow the progression of the disease in most patients for more than a few months. Although many new compounds have been tested recently, only a few have been approved for treatment. Of the several new drugs recently approved by the U.S. Food and Drug Administration (FDA), only edaravone (marketed under the trade name Radicava) has been shown to be safe, but its effectiveness was limited to patients with a disease duration of less than 2 years. This was not surprising, since it is well known that in most diseases, the timing of treatment is crucial to its effectiveness. Unfortunately, it is also known that ALS begins long before a patient experiences the first symptoms of the disease. The results of clinical trials of edaravone suggest that if the ALS phenotype could be detected early enough, treatment could more effectively delay disease progression. Moreover, this may also apply to other drugs that are currently less effective. However, defining the appropriate therapeutic window for a disease that does not manifest itself until half of the MNs have degenerated is a major challenge. There is therefore an urgent need for appropriate biomarkers that can be used to diagnose the presymptomatic stage of ALS. Recently, it has been suggested that measures of MN excitability, such as the MU firing rate at the recruitment threshold, deltaF or the duration of afterhyperpolarization, examined using noninvasive HD-sEMG arrays, may be promising candidates for ALS diagnostics (Fajardo et al., 2022). Such measurements have been described in several recent articles. The results of published works (Nishikawa et al., 2022; Noto et al., 2023; Trajano et al., 2023) shed new light on our results regarding the duration of AHP in MNs in ALS patients (Piotrkiewicz & Hausmanowa-Petrusewicz, 2011). Thus, it was confirmed that the early symptomatic stage of ALS, when there is mild muscle weakness, is different from the later stages and can be considered as the prodromal period defined in (Benatar et al., 2021). This article describes the experimental evidence supporting this finding and hypothesizes a possible evolution of MN discharge properties during the course of the disease. It also proposes a framework for future studies that could advance ALS research toward resolving the mystery of its etiology. In our study (Piotrkiewicz & Hausmanowa-Petrusewicz, 2011) we examined 20 patients diagnosed with definite (sporadic) ALS and 13 age-matched control subjects. Motor unit (MU) potentials were recorded from the left biceps brachii using fine wire electrodes during several sustained isometric muscle contractions of different constant forces. Data analysis included estimating the duration of AHP by calculating the transition interval (TI) from the plot of the absolute differences between each pair of adjacent interspike intervals, ISIs (CD2 = |ISIi+1 – ISIi |) plotted against the mean ISI of the same ISI pair (MISI2). An example of a plot of CD2 vs. MISI2 is shown in Fig. 1 (circles). Mean CD2 values, CDm, were calculated for each 10 ms bin of MISI2 (shifted every 5 ms) and were plotted against the mid-bin MISI2 value (Fig. 1, diamonds). The transition interval (arrow) was determined at the intersection of the 2 straight lines, fitted to an initial and terminal fragment of the plot of CDm vs. MISI2. The degree of muscle lesion was measured as relative force deficit (RFD), expressing the reduction in the patient's maximal voluntary contraction (MVC) as a percentage of the gender-respective mean control value. The range of RFD calculated for our patients was 0% ≤ RFD ≤ 92.6%. The most important result of our 2011 study was the relationship between TI and RFD (Fig. 2). TI was presented as the mean value of all TIs for single MUs from a given patient. Therefore, this value did not represent a single MN, but rather a pool of MNs. The relationship was approximated by 2 straight lines, which show that for RFD < 30%, the TI was practically constant and shorter than the shortest TI of the control subjects. Patients with RFD in this range were designated as Group 1. The difference between the TI values for single MUs from this group and those of the age-matched control group was statistically significant (P < 0.001). Accordingly, the MU discharge rates in Group 1 patients were higher than in the control subjects. We also distinguished Group 3 patients with RFD > 80%, whose discharge rates were lower than the control values. All differences were statistically significant (P < 0.007). The MU discharge rates of the remaining patients (Group 2) were within the normal range. One of the reviewers of our 2011 paper suggested that we should approximate the patient data with only one straight line. Nevertheless, we insisted on a 2-line approximation, believing that there may be at least two different symptomatic stages of the disease, limited by the RFD of about 30%. However, we were able to obtain mean TI for only three patients from Group 1, which did not allow us to be confident in this finding. As mentioned above, recently published studies confirmed our results by reporting signs of MN hyperexcitability in the early stages of ALS, which included shorter AHP and higher firing rates than those measured in control subjects (Nishikawa et al., 2022; Noto et al., 2023). A substantial part of the methods used in these works overlapped with our methods. According to a study by Trajano et al. (2023), hyperactivity was increased in the early stages of the disease when there was little muscle weakness and then decreased with disease progression and increasing muscle weakness. This also confirmed our other results, i.e. increased AHP duration for RFD > 30% and lower firing rates for RFD > 80% compared to control values. In Fig. 3, our data on the age-dependency on AHP duration in patients with ALS have been combined with the control data from our previous study (Piotrkiewicz et al., 2007). The graph is not as clear as the one presented in Fig. 2, since the relationship between RFD and patient age (Fig. 3B) is complicated, apparently because of differences in the rate of disease progression in different patients (note the data from the two youngest patients with the fastest disease progression). These points in Fig. 3B appear as clear outliers (open squares). The red dashed line has been drawn in Fig. 3A through most of the patient data. It reveals another outlier (open circle), which suggests that the disease progressed more slowly in this patient. If we ignore outliers, we can distinguish two symptomatic stages of ALS, stage 2, when the duration of AHP is practically constant, and stage 3, when it increases rapidly (3A, dashed line). Stage 2 corresponds to the prodromal period of mild motor impairment defined in (Benatar et al., 2021). The TI values at stage 2 are in the range of those of the youngest control participants. This could suggest that MN maturation in the initial stages of the disease was delayed until about age 50 and later transformed into a rapid ageing process. However, it is well known that in the presymptomatic stage of ALS the most sensitive MNs degenerate and their muscle fibres are reinnervated by other MNs, so it would not be reasonable to assume that these processes do not affect MN properties. MN degeneration begins at an unknown time point in the presymptomatic stage and results in an initial gradual decline in AHP duration continuing into prodromal stage 2, followed by a rapid increase in stage 3. Such a time course of MN excitability has been suggested by (Weddell et al., 2021) and confirmed by (Trajano et al., 2023). MNs innervating FF muscle fibres in animal models of ALS have been shown to be most vulnerable to degeneration. On the other hand, the duration of AHP has been shown to be matched to the twitch duration of its muscle unit (Kernell et al., 1999). At first glance, one would expect that an MN pool that loses its 'fastest' members would evolve toward a 'slower' phenotype, with a longer average AHP duration. Why, then, was a shorter AHP duration found in strong muscles? The most likely answer to this question is a trophic effect exerted by fast muscle fibres on reinnervating MNs. Indeed, there is considerable evidence that this occurs in the case of MU remodelling due to ongoing reinnervation. Kryściak et al. (2014) examined the properties of single MUs in the medial gastrocnemius muscle of the SOD1 rat model of ALS at 3 stages: asymptomatic (ALS I); early symptomatic (ALS II); and terminal (ALS III). They reported that higher proportions of FF (fast fatiguing) and S (slow) MUs were observed in ALS II, whereas higher proportions of S and lower proportions of FF MUs were noted in ALS III compared with control (wild type) animals. It was also found that 'slow' MNs that reinnervated fast muscle fibres acquired some of the properties of fast MUs, including lower fatigue resistance, greater force generation, and higher action potential amplitudes. Weddell et al. (2021) also observed that the first MUs recruited (most likely the initially slowest) developed features similar to fast twitch MUs and suggested that reinnervating MNs may adopt similar properties to those they compensate for. Further evidence for the evolution of MN properties was provided by molecular studies in the SOD1G93A mouse animal model of ALS. One of the compounds tested was osteopontin (OPN), which has been reported to be a potential ALS-specific CSF biomarker that enhances the transcription and activation of matrix metalloproteinase-9 (MMP-9). MMP-9 is known to be exclusively expressed by fast MNs and selectively conditions them to become susceptible to toxicity induced by mutant SOD1 (Kaplan et al., 2014). Morisaki et al. (2016) examined the expression of OPN in the SOD1G93A mouse model of ALS in 4 consecutive stages (60, 100, 120 and 140 postnatal days). Motoneurons were classified analogously to the classification of the muscle units they innervated, i.e. FF, fast fatigable, FR, fast resistant to fatigue, and S, slow. The study revealed 4 types of MNs expressing OPN and MMP-9, presumably representing different MN types: OPNlow/MMP-9high (FF MNs), OPNhigh/MMP-9high (FR MNs reinnervating FF fibres); OPNhigh/MMP-9low (S MNs reinnervating FF fibres), and OPNlow/MMP-9low (γ MNs). Fig. 4 shows the changes in the number (A) and percentage (B) of different types of MNs in the disease course. The authors conclude that OPN acts as a marker of FR/S-type MNs and a disease modifier that exerts selective effects on MN vulnerability. OPN is involved in the second wave of neurodegeneration in ALS, during which MMP-9 renders ALS-resistant FR/S MNs susceptible to SOD1-mediated toxicity. In summary, the molecular basis of neuronal diversity provides new insights into the mechanisms of selective vulnerability to neurodegeneration (Kaplan et al., 2014). Changes in MN properties have been observed in animal models of ALS as early as at birth. Unfortunately, there is no solid evidence for disease onset in humans, except for one article summarizing a longitudinal study of a small sample of patients with familial ALS (Aggarwal & Nicholson, 2002). In 2 of 19 SOD1 mutation carriers studied, the authors observed a sudden reduction in MU number several months before the onset of weakness. In the remaining 17 mutation carriers, normal MU numbers were maintained throughout the 3-year period. In my opinion, the evidence presented above allows me to formulate a hypothesis about the evolution of the mean AHP of the MN pool in the course of ALS. I assumed that MN degeneration starts at some point in the presymptomatic phase and results in a short initial prolongation of the AHP duration due to the reduction of the number of FF MNs. Then reinnervation begins, and reinnervating FR and S MNs gradually acquire a faster phenotype, causing a gradual decrease in the AHP duration lasting in the prodromal phase. As reinnervating MNs acquire faster phenotypes, their vulnerability to degeneration increases and their capacity for reinnervation decreases, ultimately resulting in a rapid acceleration of the disease in stage 3. Figure 5 shows the hypothetical time course of the AHP duration for most of our patients (middle curve), as well as for faster (left curve, squares) and slower progression (right curve, circle). The starting points of the curves were chosen arbitrarily. This hypothetical time course corresponds to the changes in MN types described by Kryściak et al. (2014) and Morisaki et al. (2016), as well as to the changes in excitability postulated by Bashford and Baker (2020) and confirmed by Trajano et al. (2023). The need for biomarkers that could be useful for pre-symptomatic ALS screening was recently highlighted in the article of Benatar et al. (2021), based on the results of the First International Pre-Symptomatic ALS Workshop. The results of recent studies have proven that such biomarkers can be obtained from the analysis of MU potential trains recorded using the noninvasive HD sEMG technique, i.e. measures of MN excitability (Fajardo et al., 2022) and the number of MUs, which can be estimated by several methods, the most popular of which is MUNIX. The biggest problem hindering early diagnosis is the long latency period of ALS, which prevents the identification of a group of patients at the earliest stage of the disease. It has been shown that the first symptoms appear only when the patient has already lost about half of his MNs. The assessment of the mean duration of AHP, discussed above, seems to be the most promising biomarker among the measures of MN excitability, because it is related to one of the intrinsic features of MNs, as well as to the muscle phenotype, which clearly changes during the progression of ALS. Although the very beginning of ALS cannot be determined at present, the presented hypothetical time course of the mean duration of AHP of the MN pool indicates the existence of a prodromal period of mild motor impairment in ALS, during which drugs such as edaravone can be used to effectively improve the patient's condition. ALS is a focal disease (Ravits et al., 2007), i.e. disability most often starts in one region of the body and then spreads to other regions. Therefore, if the diagnosis of ALS is made in a muscle with mild disability, there is a chance that studying MU discharge patterns in other muscles will provide some information about the presymptomatic stage of ALS (Din Abdul Jabbar et al., 2024). Currently, the only possible cohort in which MN evolution and MU counts could be studied in the presymptomatic period are patients with fALS. However, it should be remembered that in most of these patients, the disease has a very slow progression, lasting more than 10 years (Camu et al., 1999). Aggarwal & Nicholson (2002) did not report any MU loss in 17 of 19 patients with familial ALS during their 3-year study, so it is likely that much longer will be needed to obtain any significant results. Therefore, it may be difficult to find funding institutions that would offer grants for such a long-term project. Perhaps a way out of this situation would be to reach a consensus on a repertoire of biomarkers that would be measured in every ALS clinic in every patient with familial ALS. Such a consensus should also include tests for early developmental abnormalities, as suggested by Kiernan et al. (2019). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None declared. Maria Piotrkiewicz: Conception or design of the work, drafting the work or revising it critically for important intellectual content, final approval of the version to be published and agreement to be accountable for all aspects of the work. None.