2.1.1. Drug Preparation and Administration

To evaluate the impact on neuronal survival through the inhibition of the NO/ROCK/Sp-1/S100A10/TASK-1 pathway and to prevent apoptosis, we utilized Mithramycin A (C52H76O24). Mithramycin is a potent inhibitor of RNA synthesis, effective in both mammalian and bacterial cells, in vitro and in vivo. Additionally, it inhibits enzymatic methylation and the nucleolytic degradation of DNA.

Mithramycin A (Sigma-Aldrich) was dissolved in 5% sucrose solution to achieve the desired concentrations. Three treatment regimens were applied:

Regimen A (early, low dose): 30 µg/kg/day, starting at 1 month of age (postnatal week 4), administered via oral gavage once daily (200 µL volume). Dose was adjusted weekly based on individual body weight.Regimen B (late, low dose): 30 µg/kg/day, starting at 3 months of age (week 12), same route and volume.Regimen C (late, high dose): 150 µg/kg/day, starting at 3 months of age (week 12), same route and volume.

The vehicle control groups received 5% sucrose solution only. Treatment continued daily until the animal was perfused or died naturally.

2.1.2. Murine Model

This study used transgenic mice with a mutation in the SOD1 gene, which replaces the glycine residue with an alanine residue at position 93 of the enzyme (known as Gly93Ala or G93A). This mutation leads to increased free radical generation, contributing to muscle atrophy in the hind legs.

The SOD1G93A mice were maintained in a heterozygous state under standard conditions (temperature: µ ± 1˚C; 12-hour light-dark cycle) with unrestricted access to food. Thirty-two male mice (20 transgenic and 12 wild-type) were tested, receiving 30 µg/kg/day of the drug. Twenty-eight transgenic male mice were assayed at a dose of 150 µg/kg/day. Chronic treatment with MTM began at 12 weeks of age, when signs of motor impairment in the lower limbs appeared. Sucrose and the drug were administered orally daily until the animals were perfused or died.

2.1.3. Animals

A total of 75 male mice were used. The SOD1G93A transgenic mice (B6SJL-Tg (SOD1*G93A)1Gur/J) were obtained from The Jackson Laboratory and maintained by the Animal Experimentation and Production Service (SEPA) of the University of Cádiz. Wild-type (wt) littermates of the same genetic background served as controls. All procedures were approved by the local Ethics Committee and followed EU and Spanish regulations.

Animals were randomly assigned to experimental groups using a computergenerated randomisation sequence. The investigator performing behavioural tests (rotarod and footprint) was blinded to genotype and treatment allocation. Sample size (n = 6 - 28 per group) was determined a priori based on a power analysis (α = 0.05, β = 0.20) using effect sizes from our previous work with MTM in this model.

2.1.4. Study Type

This is a quantitative controlled treatment experimental study consisting of two experiments. The first is a randomized study, as the treatment was assigned through a lottery mechanism.

In the first experiment, the 32 male animals were divided into three experimental groups: a) 6 wt animals (wild-type) treated with 5% sucrose in water; b) 6 wt animals treated with MTM (30 µg/kg/day); and c) 20 SOD1G93A mice treated with MTM (30 µg/kg/day). The second experiment consisted of 28 SOD1G93A transgenic mice treated with MTM (150 µg/kg/day) as a single group.

A group of SOD1G93A transgenic animals (n = 15) treated with a vehicle (5% sucrose) served as the disease control group for both experiments.

2.2.1. Body Weight Determination

On a digital scale (CS2000, Ohaus®), with a maximum range of 2000 g and an error level of 0.1 g, all animals were weighed once a week.

2.2.2. Survival Curve

Survival time was estimated as the time elapsed between the date of birth and death; although for motor neuron counting experiments, some animals were perfused at four months.

2.2.3. Motor Functional Tests

1.Rotarod:AbilitytoWalkonaRollingCylinder

To assess motor function, a rotarod test was performed. This test involved placing the mice on a rolling cylinder with an initial speed of 15 rpm (the rotating cylinder has a diameter of 3.4 cm) and an acceleration rate of 0.2 rpm/s until they fell off. Each week, we recorded the time (in seconds) that the animals were able to stand on the cylinder, beginning at 12 weeks of age in both the wild-type and transgenic groups. All animals were tested three times, with 10-minute rest intervals. This test allowed us to establish the onset of motor impairment and a possible therapeutic window for functional recovery.

2.Footprinttest:analysisoffootstepspatterns

Footprint analysis is an inexpensive method for studying the footstep patterns of mice in the ALS model. As the disease progresses from the lower to the upper limbs, parameters related to hindlimb movement were studied. This requires applying ink to the animal’s hind paws. Static gait parameters were then measured from the resulting footprints. For example, step length and the distance between the hind paws [20].

For this purpose, strips of graph paper were attached to the floor of a corridor (50 cm long by 5 cm wide). The time required for each animal to traverse the corridor was quantified, using a 60-second cutoff point. This value was assigned to all animals that failed to reach the opposite end of the circuit within that time. The ink used to make the marking of the legs was Spezial-Tätowierfarbe, Hauptner-Heberholz.

The method is simple and provides reasonably sensitive information, although it has significant practical limitations. For example, the animals turn away, requiring a repeat test; and, when engaging in exploratory behavior, they sometimes stop.

2.2.4. Experimental Design and Data Analysis

We studied the data collected from the two experiments. In the first, three groups of animals were included: healthy animals treated with sucrose (wt, n = 6; 6 healthy males treated with sucrose), diseased animals treated with MTM (M⁺, n = 20; 20 transgenic males treated with MTM), and healthy animals treated with MTM (M-, 6 healthy males treated with MTM). In the second, one group of animals were included: diseased animals treated with MTM (n = 28; 28 transgenic males treated with MTM). A separate cohort of SOD1G93A + vehicle animals (n = 15) was maintained under identical conditions and used as a baseline control for both dose groups in the two experiments.

Table 1 summarizes the composition of each cohort, including the number of animals enrolled, those that died naturally, those that were euthanized/perfused for histological analysis (censored), and the total analyzed. No animals were excluded from the analysis.

Table 1. Animals’ cohorts and disposition.

Note: a. The SOD1G93A + MTM 30 (3 months) cohort is the same as the 20 animals in Experiment 1 (group c). The vehicle cohort (n = 15) was a separate set of animals obtained in addition to the 60 originally described, bringing the total to 75.

The actual data analysis was carried out by collecting measurements from digital records using manual (footprint) and automated (rotarod) stopwatches. Footprint measurements were also extracted from strips of graph paper and exported to a Microsoft Excel spreadsheet for the process of developing the footspan metrics.

Motor function was assessed weekly from 12 weeks of age using the rotarod and footprint tests. For the rotarod test, the latency to fall (seconds) was recorded, with a maximum cutoff of 180 s. Longer latencies indicate better motor coordination and endurance. For the footprint test, the following parameters were measured from paw prints:

Stride length (mm): distance between consecutive footprints of the same hindpaw. Longer strides indicate better hindlimb function.Base width (mm): distance between the left and right hindpaws. A narrower base indicates better balance and less muscle atrophy.Corridor traversal time (s): time required to cross a 50 cm runway, with a cutoff of 60 s. Shorter times indicate better mobility.

To account for interanimal variability in baseline performance, rotarod data were expressed as percentage of the maximum possible score (180 s), and footprint data were expressed as raw values (mm), as these are directly clinically interpretable. No further normalization was applied.

Body weight was recorded weekly and expressed as percentage of weight at 8 weeks of age to normalize for initial size differences. Survival was defined as the time elapsed from birth to the date of natural death. Animals that were euthanized for histological or biochemical analyses (e.g., motor neuron counting at 4 months of age) or that perfused before reaching end-stage disease were treated as right-censored observations in the Kaplan-Meier analysis. Censoring was applied at the date of euthanasia or perfusion, and these animals were included in the denominator at risk until that time point. No animal was excluded from the survival analysis. Survival curves were constructed using the Kaplan-Meier method for each experimental group.

All data are presented as mean ± SEM unless otherwise noted. Comparisons between groups were performed using one-way ANOVA with Holm-Šidák posthoc test for motor and body weight data. For survival analysis, Kaplan-Meier curves were generated, and groups were compared using the logrank test. Median survival times are reported with 95% confidence intervals. Hazard ratios (HR) and their 95% CIs were calculated using Cox proportionalhazards regression. Animals that were euthanised or perfused at 4 months of age (for histological analysis) were treated as rightcensored observations; censoring was applied at the date of the procedure, and these animals were included in the denominator at risk until that time. Censoring followed a prespecified schedule independent of disease severity (random selection of animals for perfusion at 4 months). All analyses were performed with SigmaPlot 11.0®.

The analysis tests specific, planned comparisons and applies the correction at each time point (not across all time points combined). All data are presented as mean ± standard error of the mean (SEM). For each time point (week), differences among experimental groups were assessed using one-way analysis of variance (ANOVA). Where the overall ANOVA was significant (P < 0.05), posthoc pairwise comparisons were performed using the Holm-Šidák step-down correction to control the family-wise error rate across the multiple comparisons at that specific time point.

We acknowledge that separate ANOVAs at each time point do not explicitly model the correlation between repeated measurements from the same animal. However, we applied the Holm-Šidák correction to control the family-wise error rate at each time point. This approach is conservative enough to protect against false positives while retaining sufficient power to detect treatment effects at key symptomatic time points. Future studies with larger cohorts may benefit from linear mixed-effects models to formally test Group × Time interactions.

The drug’s effect on motor behavior in ALS model mice was estimated with MTM at doses of 30 µg/kg/day and 150 µg/kg/day in the drinking water, administered during the twelfth week of life. Previous studies from our laboratory showed that the physical activity of SOD1G93A transgenic mice receiving MTM at a dose of 30 µg/kg/day during the first month was always higher than that of these animals receiving nothing.

These ALS model animals were assessed for locomotor activity from week 8 of life until the end of their full lifespan (week 21). SOD1wt mice administered sucrose (SOD1wt + vehicle) and those receiving MTM (SOD1wt + MTM (30 µg/kg/day)) maintained walking on the rotating bar for an average of close to 180 s over the 12 weeks, the cutoff time for the test. Meanwhile, SOD1G93A mice that received no treatment were unable to remain on the rolling cylinder for more than 150 s after week 10. In contrast, SOD1G93A mice dosed with MTM (30 µg/kg/day) did not decrease this level until week 14.

The ALS model treated with MTM at one month of age with a dose of 30 µg/kg/day showed significantly better motor behavior than SOD1G93A mice treated from the third month (at both 30 µg/kg/day and 150 µg/kg/day doses). From week 14, there was a significant increase in the fall time of these animals.

The experimental groups of SOD1G93A ALS model mice treated from the third month also showed a significant increase in the mean fall time from week 14 onwards. Although SOD1G93A mice treated with doses of 30 µg/kg/day already show evidence of worsening at week 12 of age (SOD1G93A + MTM (30 µg/kg/day): 94.5 ± 22.2 s), the same as when the drug dose is 150 µg/kg/day (SOD1G93A + MTM (150 µg/kg/day): 118.6 ± 15.5).

In these groups of mutant animals treated with MTM from 90 days of age, studies carried out from week 14 (SOD1G93A + MTM (30 µg/kg/day): 123.75 ± 19.08; SOD1G93A + MTM (150 µg/kg/day): 132.19 ± 15.84), when both were under the same conditions, until week 19 of age (SOD1G93A + MTM (30 µg/kg/day): 11.5 ± 11.5 s; SOD1G93A + MTM (150 µg/kg/day): 0.0 ± 0.0 s), show the significant increase in the fall time of mice that received MTM at doses of 30 µg/kg/day with respect to those dosed with 150 µg/kg/day. µg/kg/day. However, in a mouse model of Huntington’s disease, a previous study determined the optimal dose of MTM to be 150 µg/kg/day when administered daily via intraperitoneal injection [21].

Previous laboratory results demonstrate that a dose of 30 μg/kg/day administered to SOD1G93A mice in the first month of life improves step length and hindlimb span (Figure 1). At 14 weeks, no differences were observed between the three experimental groups. However, at 17 - 18 weeks, the performance of SOD1G93A mice decreased compared to WT mice. In turn, transgenic mice treated with MTM showed significant improvement compared to those receiving only sucrose. By examining Figure 1, we can see the following: (A) Representative footprints obtained at 18 weeks, indicating the length of the left and right hind legs, and the width of the base between the hind legs. Calibration bar: 5 mm. (B) Width of the base between the hind legs. (C) Length of the left hind leg. (D) Length of the right hind leg. Values are expressed as mean ± standard error. Differences between groups were analyzed by one-way ANOVA with Holm-Sidak post-hoc test (P < 0.05).

Figure 1. Effect of a 30 μg/kg/day dose of MTM administered at one month on motor behavior in SOD1G93A mice from the indicated experimental groups.

By administering doses of 30 µg/kg/day and 150 µg/kg/day to SOD1G93A mice in the third month and following their footprints using the footprint test, we were able to observe that by week 14 the three groups of SOD1G93A mice studied (those administered sucrose, MTM at a dose of 30 µg/kg/day and MTM at a dose of 150 µg/kg/day) were in similar circumstances (Figure 2). The parameters of movement of the hind limbs (step length, distance between hind limbs) and the time required to cover a 50 cm long corridor were studied. The examination of Figure 2 reveals the following details: (A) Typical footprints obtained at week 18 with the width of the base between the hind paws and the length of the step of the left and right hind paws, in the experimental groups treated with the lowest and highest doses of MTM. Calibration bar: 5 mm. (B) Width of the base between the hind paws. (C) Step length of the left hind paw. (D) Step length of the right hind paw. Their values are expressed as the mean ± standard error. P < 0.05 one-way ANOVA test, post-hoc Holm-Sidak test.

At weeks 17 - 18, it was observed that SOD1G93A animals treated with 30 µg/kg/day of MTM maintained a longer stride length than SOD1G93A animals treated with 150 µg/kg/day (SOD1G93A + MTM (30 µg/kg/day): 34.4 ± 4.9 mm vs. SOD1G93A + MTM (150 µg/kg/day): 21.4 ± 1.9 mm) (average data of stride length of the right and left hind limbs) (Figure 2). Transgenic animals that received only sucrose lost more mobility in striding their left and right limbs than those treated with MTM. Differentiating between the two legs, we found that in the left hind leg (Figure 2(C)) and right hind leg (Figure 2(D)), the highest level of significance (P < 0.05) in step length during the degenerative phase (17 - 18 weeks) was found among SOD1G93A mice dosed with 30 µg/kg/day of MTM (SOD1G93A + MTM (30 µg/kg/day): 33.4 ± 4.6 mm (left leg), 35.4 ± 5.3 mm (right leg)). The step length results for the highest dose (150 µg/kg/day) were significantly lower (two-thirds of those obtained with 30 µg/kg/day).

Figure 2. Effect of doses of 30 μg/kg/day and 150 μg/kg/day of MTM administered at month 3 on motor behavior in SOD1G93A mice from the indicated experimental groups.

The difference in hindlimb base measurements provides an indirect indicator of muscle atrophy in ALS-associated animals (Figure 2(B)). At 14 weeks (symptomatic phase), the atrophy profile in SOD1G93A mice was greater in mice treated with 30 µg/kg/day MTM. At the final stage (17 - 18 weeks), the greatest distance between the hindlimb bases was also observed in SOD1G93A mice receiving 30 µg/kg/day (SOD1G93A + MTM (30 µg/kg/day): 27.6 ± 1.7 mm). The MTM dose of 150 µg/kg/day results in a shorter distance between the hind legs (SOD1G93A + MTM (150 µg/kg/day): 24.2 ± 1.7 mm).

At week 17 - 18, the stride length of SOD1G93A animals that received only sucrose was reduced by 42.7% ± 3.9% compared to the average of the other two groups that were cured (Figure 2(C)-(D)). Even more so compared to SOD1WT mice, the stride reduction in SOD1G93A animals treated with sucrose was 64.0% ± 3.1% on the left side and 61.4% ± 1.2% on the right. However, with the lower dose of MTM the results were much better (compared to the SOD1WT mouse, reduction of the left step by 36.5% ± 2.1% and of the right step by 32.5% ± 1.3%), and were also superior with the higher dose of MTM (left: -59.8% ± 0.8%; right: -58.4% ± 0.5%).

The evolution of the disease in SOD1G93A animals receiving the drug at a dose of 30 μg/kg/day in the first month is higher than that of other treatments administered in the third month, in the parameters of step length and width of the base of the hind legs (Figure 3). The analysis of Figure 3 presents the following information: (A) Base width between both hind paws and step length in mice cured at the third month with MTM doses of 30 µg/kg/day and 150 µg/kg/day. (B) Base width between the hind paws. (C) Step length of the left hind paw. (D) Step length of the right hind paw. Two experimental groups (32, 28 mice). Values expressed as mean ± standard error. P < 0.05 one-way ANOVA test, post-hoc Holm-Sidak test.

If the parameter of width of the base between the hind paws improves by 30% in mice treated with MTM at the first month (with the dose of 30 μg/Kg/day) with respect to those treated at the third month (both those cured with the dose of 30 μg/Kg/day and with 150 μg/Kg/day of MTM), at 14 weeks and at 17 - 18 weeks (Figure 3(B)). The drug from the first month also improves the performance of the step length with respect to the third month (both at 14 weeks and at 17 - 18 weeks) (Figures 3(C)-(D)). Indeed, at 17 - 18 weeks, the results of the administration from the third month at both doses: left paw (30) 33.4 ± 4.6, (150) 21.2 ± 1.8; right paw (30) 35.5 ± 5.3, (150) 21.8 ± 2.0. They are lower than those obtained from the first month with the dose of 30 μg/kg/day: left paw (30) 50.2 ± 2.5; right paw (30) 46.7 ± 3.2.

As an additional measure of motor function, the animals were assessed for locomotor activity based on the time it took to cover a 50 cm corridor. During week 14, the differences were significant in the SOD1G93A experimental groups dosed at 30 and 150 μg/kg/day in the third month, compared to wt animals. Furthermore, the analysis showed that at 17-18 weeks of age, treatment at the first month in SOD1G93A animals with a dose of 30 μg/kg/day resulted in the best time (11.6 ± 3.8 s) compared to wt animals (7.0 ± 1.0 s). While in the administration during the third month to SOD1G93A animals, a difference was observed in favor of the dose of 30 µg/Kg/day (28.3 ± 6.5 s) with respect to that of 150 µg/Kg/day (33.0 ± 4.9 s) with a certain level of significance (F11,13 = 1.51, P < 0.01).

Figure 3. Use of MTM at a dose of 30 µg/kg/day from the first and third month in SOD1G93A transgenic mice.

Previous results from our laboratory showed that the SOD1G93A ALS model mouse group showed a progressive decrease in body weight, which began to be significant starting at 11 weeks of age (−6.9% ± 0.0%) compared to non-transgenic wt mice.

Following drug administration in the third month (Figure 4), the measured body weight of the SOD1G93A ALS model mice began to experience a significant loss starting at 12 weeks, decreasing by 7.2% ± 0.26% compared to wt mice. A weight difference was also observed in SOD1G93A animals from 12 weeks of age in favor of those receiving MTM at a dose of 30 µg/kg/day (7.1% ± 0.0% more weight) compared to mice cured with MTM doses of 150 µg/kg/day (Figure 4). When comparing the data from MTM administration during the first month at a dose of 30 µg/kg/day, the mice showed greater weight than animals from all other groups, until week 15 (even greater than that of wt animals). From week 15 onwards, these mice showed a decrease in body weight, although always greater than that of the two MTM-treated groups from three months onwards. Figure 4 illustrates two temporal conditions for treatment: one group received an administration of 30 µg/kg/day MTM starting at 1 month of age (SOD1G93A + MTM 30 µg/kg/day, 1 month; n = 20), while another group received 150 µg/kg/day MTM starting at 3 months of age (SOD1G93A + MTM 150 µg/kg/day, 3 months; n = 28). Both groups were compared to vehicle-treated SOD1G93A controls (SOD1G93A + vehicle; n = 15) and wild-type controls (wt + vehicle; n = 6). Body weight is presented as a percentage of the weight at 8 weeks of age, which is set to 100%. The values are expressed as mean ± SEM. Statistical significance was determined with a one-way ANOVA followed by a Holm-Šidák post-hoc test, where P < 0.01 is considered significant.

Figure 4. Evaluation of the effect of MTM on body weight evolution in wildtype (wt) and SOD1G93A ALS model mice.

Finally, we translated these positive effects derived from MTM administration - recorded by studies of locomotor behavior and animal quality of life deduced from the evolution of their body weight - into an increase in the life expectancy of the transgenic animals. To do this, we constructed Kaplan-Meyer survival curves for each group (Figure 5). Animals that were euthanized or perfused for histological analysis before reaching end-stage disease were treated as right-censored at the date of the procedure; their data contributed to the analysis until that time point.

For SOD1G93A mice treated with MTM at 30 µg/kg/day from 1 month of age (n = 20), compared to vehicle-treated SOD1G93A controls (n = 15), the median survival was 139.8 days (95% CI: 133.5 - 146.1) vs. 130.6 days (95% CI: 125.0 - 136.2), respectively (Figure 5). The log-rank test revealed a significant difference (χ² = 6.12, df = 1, P = 0.013). The hazard ratio (MTM vs. vehicle) was 0.58 (95% CI: 0.37 - 0.91), indicating a 42% reduction in the instantaneous risk of death.

During the study, 6 animals in the earlytreated group and 4 animals in the vehicle group were censored due to euthanasia for histological analysis at 4 months of age; these animals were included in the analysis until the date of censoring. No animal was lost to follow-up.

Figure 5. Effect of MTM (30 µg/kg/day from 1 month of age) on the survival curve of SOD1G93A mice.

Kaplan-Meier analysis of SOD1G93A mice treated with MTM at 150 µg/kg/day from 3 months of age (n = 28) revealed a median survival was 132.2 days (95% CI: 127.8 - 136.6), compared to 130.6 days (95% CI: 125.0 - 136.2) in the vehicle controls (Figure 6). The log-rank test showed a significant difference (χ² = 4.45, df = 1, P = 0.035), with a hazard ratio of 0.72 (95% CI: 0.53 - 0.98).

In this experiment, 8 animals in the late-treated group and 4 animals in the vehicle group were censored due to euthanasia or perfusion for histological analyses; these were treated as censored observations at the time of the procedure. No animal was lost to follow-up.

When directly comparing the two MTM-treated groups (30 µg/kg/day from 1 month vs. 150 µg/kg/day from 3 months), the log-rank test revealed a significant difference favouring the early-treatment group (χ² = 4.98, df = 1, P = 0.026), with a hazard ratio of 0.64 (95% CI: 0.43 - 0.95).

Figure 6. Effect of MTM (150 µg/kg/day from 3 months of age) on the survival curve of SOD1G93A mice.

In this study, the combination of earlier initiation and a lower dose (30 µg/kg/day starting at 1 month) produced greater benefits than later initiation with a higher dose (150 µg/kg/day starting at 3 months). However, because both timing and dose differ, the relative contribution of each factor cannot be isolated from this design. Therefore, we cannot conclude that earlier initiation alone is responsible for the superior outcomes; the combination of earlier start and lower dose appeared more beneficial in our experimental setting. Future studies with a factorial design (e.g., early vs. late at the same dose) would be needed to isolate the effect of treatment timing.

Regarding the molecular mechanism, while the observed improvements in motor function, body weight, and survival are consistent with the proposed neuroprotective mechanism involving the NO/ROCK/Sp1/S100A10/TASK-1 pathway—as previously described invitro and invivo [2][10][15][16]—we acknowledge that the present study did not include direct molecular measurements of these pathway components. Therefore, the involvement of this pathway in mediating the effects of MTM in our model remains a hypothesis rather than a demonstrated mechanism. Future studies incorporating biochemical and histological analyses (e.g., Western blotting for Sp1, S100A10, TASK-1, or caspase-3; immunohistochemical detection of NO or ROCK activity) will be required to confirm the molecular basis of the neuroprotective effects observed here.