Trimetazidine, a promising drug for amyotrophic lateral sclerosis, modulates Ca2+ influx in spinal neurons

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The mechanism underlying the effect of TMZ on locomotor activity has been proposed to rely on its ability to enhance metabolic efficiency with a consequent improvement of myogenesis and of neuromuscular junction (NMJ) and muscle function. However, although promising and therefore under clinical trials, the mechanism of action of TMZ has not been clearly disclosed; here we hypothesized that it might involve the modulation of neuronal Ca 2+ flows. We studied the effect of TMZ on Ca 2+ dynamics in vivo , by using the transgenic zebrafish line Tg(neurod1 :GCaMP6f ) in which the neuronal expression of the Ca 2+ indicator GCaMP allows to visualize Ca 2+ dynamics in neurons of zebrafish larvae. By this elegant tool, we demonstrated, for the first time, that TMZ promotes Ca 2+ influx in zebrafish spinal neurons likely enhancing motor neuron firing, which correlates with enhanced motor performance by this drug. Even though elevated intracellular Ca 2+ levels have often been associated to neurotoxicity, it is unclear if the neuronal excitability features in ALS are compensatory or pathological. Therefore, TMZ might potentially contribute to counteract neurodegeneration by modulating neuronal Ca 2+ fluxes, and transiently and selectively enhancing motor neuron firing, as well as NMJ and locomotor function, without increasing the overall neuronal excitability. This further supports TMZ repurposing for the treatment of ALS and of other conditions characterized by NMJ impairment, such as aging. Health sciences/Neurology/Neurological disorders/Motor neuron disease/Amyotrophic lateral sclerosis Biological sciences/Neuroscience/Motor control/Neuromuscular junction Biological sciences/Neuroscience/Motor control/Spinal cord Biological sciences/Physiology/Ageing Biological sciences/Drug discovery/Target identification Amyotrophic lateral sclerosis Trimetazidine Aging Spinal cord Skeletal muscle Motor neuron disease NMJ Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Trimetazidine (TMZ) is a antianginal beneficial on ischemic injury 1 . Also, it has more recently been demonstrated that TMZ acts on skeletal muscle by promoting myogenesis, by improving muscle quality and neuromuscular communication, and by increasing muscle strength in mice models of aging and cachexia, as well as in patients with peripheral arterial disease 2 – 5 . Moreover, we have reported that orally administered TMZ leads to motor performance improvement in transgenic SOD1 G93A mice, the standard animal model for Amyotrophic Lateral Sclerosis (ALS) 6 . TMZ is a piperazine derivative inhibiting the 3-ketoacyl coenzyme A thiolase which catalyzes long-chain fatty acid β-oxidation, thus leading to a shift from fatty acid to glucose as preferential used substrate. This metabolic shift has been found to optimize energy production 7 – 9 . However, evidence for this mechanism is controversial and since, based on its preclinical efficacy, TMZ is under clinical trial (NCT04788745), elucidating the details of its action is a high-priority. ALS is a neurodegenerative disease affecting both upper corticospinal motor neurons and lower ones in brainstem and spinal cord. ALS progresses from weakness to gradual muscle atrophy and inability to perform movements, till complete paralysis and death 10 – 13 . Etiopathogenesis of ALS is poorly understood and, also for this reason, it currently lacks effective treatment. Also prior to clinical symptoms, electively vulnerable motor neurons, responsible for voluntary muscle contraction, show early pathological signs including axonal transport defects, protein aggregation, mitochondrial dysfunctions and altered intrinsic excitability 14 , 15 . It has been proposed that motor neuron electrical signaling initially increases during the early stages of ALS and then it gradually decreases, as neurons degenerate, and lose their ability to properly respond to stimuli 16 – 19 . Notably, both neuronal hyperexcitability and hypoexcitability have been observed in ALS; however, it is not clear if they are compensatory or pathological events 20 , 21 . Also, disassembly and denervation of the neuromuscular junction (NMJ) occur in ALS before motor neuron degeneration, with episodes of reinnervation interpreted as attempts to regenerate NMJ 22 . Since among the mechanisms proposed to explain the origin of ALS are mitochondrial and metabolic dysfunctions 23 – 25 , we have recently focused our attention on evaluating and proving the pre-clinical efficacy of the metabolic modulator TMZ in counteracting ALS progression 6 . However, its mechanism is controversial and, in addition, we had previously observed that murine skeletal muscles treated with TMZ ex vivo display a very quick shift towards a slow-twitch contractile phenotype 3 . This very rapid effect might not be explained by gene expression modulation therefore we hypothesized an ability of this drug to modify ionic fluxes. To address this issue, we focused our attention on Ca 2+ since it is the final ionic effector of both skeletal muscle contraction and motor neuron firing. This choice was also supported by previous studies demonstrating the ability - not clearly elucidated- of TMZ to act on Ca 2+ homeostasis in cardiomyocytes 26 – 29 . To study the role of TMZ on Ca 2+ dynamics in neurons and neuronal activity in vivo , we took advantage of a model represented by the transgenic zebrafish line Tg(neurod1 :GCaMP6f ) for in vivo Ca 2+ imaging in neurons 18 . By this tool we visualized Ca 2+ dynamics in neuronal cells demonstrating the ability of TMZ to induce Ca 2+ influx and spinal neuron firing which correlates with zebrafish motor performance stimulation by this drug. RESULTS TMZ improves locomotor performance in zebrafish larvae In order to confirm the effect of TMZ on locomotion also on zebrafish larvae, which we used as model to evaluate Ca 2+ fluxes, we first performed a toxicity assay using increasing concentrations of TMZ, specifically 25 µM, 50 µM, 100 µM, 200 µM, 1000 µM and 2000 µM. We administered the drug to 4 dpf larvae and we kept them in these solutions for 24h. We observed that 1000 µM and 2000 µM TMZ were toxic for the larvae which died a few hours upon drug administration. For this reason, we performed the behavior locomotor assay by administrating 25 µM, 50 µM, 100 µM and 200 µM TMZ to 4 dpf larvae to obtain the velocity and the distance travelled by the larvae pre- and post-TMZ treatment (Fig. 1 ). Our data confirm that there is a statistically significant improvement of the locomotor performance upon both 100 µM and 200 µM TMZ treatment (Fig. 1 A, B). These data are in line with the results obtained by treating the Tg SOD1 G93A mouse model of ALS and that of aging with TMZ which increased their locomotory activity 2 , 6 and make zebrafish larvae a suitable model to unravel the mechanisms underlying TMZ effect on locomotion. Ca 2+ influx in spinal cord is stimulated by TMZ Based on the analysis of zebrafish locomotor behaviour upon TMZ treatment, along with the previous observation that ex vivo administration of 100 µM TMZ is able to act very quickly on the contraction rate of excised tibialis anterior murine muscles 3 , we hypothesized that this metabolic modulator might be able to act on skeletal muscle contractility and, consequently, on locomotion, by modifying ionic fluxes. Therefore, we decided to evaluate the firing of the skeletal muscle-stimulating motor neurons through the analysis of Ca 2+ fluxes in vivo . We used the transgenic zebrafish line Tg(neurod1 :GCaMP6f ) based on the neurod1 promoter-mediated neuronal expression of the GFP-based Ca 2+ indicator GCaMP6f . 100 µM TMZ was administered to 4 dpf Tg( neurod1 :GCaMP6f) larvae and Ca 2+ imaging was first performed on spinal cord (Fig. 2 ; 40X magnification). The images were acquired before and after TMZ treatment with the timing described in Fig. 2 A. By comparing the fluorescence signal derived from GCaMP activation before and after TMZ stimulation, a significant increase in Ca 2+ influx- and thus neuron firing- was observed upon TMZ administration (see the representative images in Fig. 2 B and video recordings in Supplementary Video S1). Accordingly, the ∆F/F0 ratio over time shows a higher fluorescent signal recorded following TMZ treatment (Fig. 2 C; POST-TMZ) compared to the signal obtained from the untreated larvae (Fig. 2 C; PRE-TMZ). The statistical analyses confirms that TMZ was effective in increasing Ca 2+ influx in neurons of the spinal cord where motor neurons are localized (Fig. 2 D). Also spinal interneurons modulating motor neuron activity are highlighted in this Tg model, therefore, by this experiment it is not possible to precisely identify the neuronal population targeted by TMZ which might likely be represented by motor neurons (indeed is it possible to observe the axon leaving the spinal cord, which indicates that motor neurons are involved; see Fig. 2 B and Supplementary video S1) or by interneurons modulating motor neuron activity. TMZ does not induce an overall brain increase of Ca 2+ influx In order to evaluate if the effect of TMZ was specific to the spinal cord or was occurring in all CNS neurons, we visualized whole brain activity by recording Ca 2+ imaging at lower magnification (4X). The images were acquired before and after TMZ treatment with the timing described in Fig. 2 A. Differently from what we had previously observed in the spinal cord (Fig. 2 ), no increase in the whole brain Ca 2+ influx was observed after TMZ treatment; see representative images (Fig. 3 A, B), videos (Supplementary video S2) and graph (Fig. 3 C). However, we decided to focus more specifically on the hindbrain region where neurons contacting and modulating motor neurons are localized, in order to get information on the kind of neurons targeted by TMZ which might be directly targeted motor neurons or spinal interneurons or also higher center neurons controlling locomotor movements. For this reason, the same magnification used for spinal cord analysis (40X) was used for the hindbrain recordings. The region chosen for the analysis is an area surrounding the Mauthner cells within the hindbrain (defined by a dotted line in Fig. 4 A). This analysis revealed an increase of the fluorescence signal in the hindbrain region of Tg( neurod1 :GCaMP6f) larvae upon TMZ administration (see the Supplementary video S3 and the representative ΔF/F0 graph in Fig. 4 B), which is, however, not statistically significant (Fig. 4 C). Nevertheless, although not reaching the significance, a relevant trend to an increase of Ca 2+ influx following TMZ treatment was recorded (Fig. 4 B and 4 C). Considering that the Mauthner cells region is involved in fish escape response, this suggests that TMZ might also act on hindbrain neurons involved in the upstream motor circuitry controlling motor neurons. DISCUSSION This study presents, to our knowledge, the first compelling evidence that TMZ significantly enhances Ca²⁺ influx in spinal neurons, indicating a potential role for this drug in modulating Ca²⁺ dynamics. This modulation may help explain the enhanced locomotion parameters induced by TMZ on aging and ALS mouse models 2 , 6 , and confirmed in this study on zebrafish. In the context of ALS, where motor neuron degeneration and progressive loss of muscle function occur 30 , TMZ's ability to improve locomotion is noteworthy. However, it is well-documented that elevated intracellular Ca 2+ levels contribute to neurotoxicity, and that altered Ca 2+ homeostasis is a pathological feature of ALS 31 , 32 . On the other hand, even though hyperexcitability and fasciculations are features of ALS 33 – 37 and therefore increased excitability triggered by TMZ might, at first glance, be considered detrimental, some issues need to be considered. First of all, hyperexcitability and fasciculations origin and contribution to the pathophysiology of ALS are unknown. It has been proposed that proximal and distal factors exist; intrinsic excitability of motor neurons, supraspinal excitability from upper motor neurons and altered regulation by interneurons, Renshaw cells or propriospinal neurons -possibly associated to excitotoxic synaptic milieu likely due to altered glutamate levels. Also, it has been suggested that, with the progressive dysfunction of lower motor neurons, fasciculations arise in abnormal, reinnervated motor units; then, as motor neurons degenerate, fasciculations become less prominent. Fasciculations might therefore occur along with compensatory -but insufficient- reinnervation, thus possible being early symptoms of aberrant firing of motor neurons 37 , and, therefore, hyperexcitability might not necessarily be the cause of neurodegeneration. In this scenario, it can be hypothesized that drugs triggering Ca 2+ influx, spinal neuron excitability and consequent skeletal muscle contraction might be compensatory and aiming at counteracting neurodegeneration and avoiding NMJ denervation. This is in accordance with our previous finding that TMZ protects against NMJ impairment 6 . Another important issue worth to be discussed concerns the occurrence of hypoexcitability in spinal motoneurons of ALS mouse models 20 , 21 , 38 – 41 , whereas cortical hyperexcitability has been described as an early feature in ALS thought to contribute to neuronal stress and subsequent degeneration 42 – 46 . Moreover, it has been reported that motor neurons derived from induced pluripotent stem cells obtained from ALS patients exhibit hyperexcitability and/or hypoexcitability 19 , 33 , 47 – 51 , and that motor neurons recorded from ALS patients are hypoexcitable 52 . Based on some author’s hypothesis, motor neurons do not develop hyperexcitability before NMJ denervation, but some motor neurons fails to produce sustained firing, and hypoexcitability occurs, despite the fact that their NMJs are still functional 38 , 39 . It has been proposed that hypoexcitability might lead to peripheral disconnection and to reduced functionality of fast fatigable NMJ and that hyperexcitability might arise in the remaining resistant neurons and compensate for the hypoexcitable vulnerable ones 53 . In this view, hypoexcitability might be the earliest pathological sign of some motor neurons -possibly due to the accumulation of misfolded SOD1 proteins- leading to dysfunctional motor units, whereas hyperexcitability might be neuroprotective and compensatory 20 , 38 , 39 , 54 . To sum up, despite the discussed hypothesis, it is still obscure whether hyperexcitability and hypoexcitability are compensatory or represent pathological traits in ASL. Therefore, we believe that the potential beneficial therapeutic action of TMZ, a drug transiently enhancing motor neuron excitability, protecting against the dismantlement of NMJs and improving locomotory abilities 6 deserves consideration and a thorough investigation. Further investigation is needed to identify the specific spinal neuronal population targeted by TMZ; indeed, we cannot distinguish motor neuron somata from large spinal interneurons modulating motor neuron activity, even though the observation of the axon leaving the spinal cord (Fig. 2 B; Supplementary video S1) indicate that motor neurons are involved. To elucidate this issue, transgenic zebrafish lines for in vivo Ca 2+ imaging in specific spinal neuron populations would be helpful. These data also reveal an interesting specificity of TMZ effect, which selectively affects spinal neurons, thus suggesting a particular effectiveness in modulating motor control. In ALS, where the specific motor neuronal population is affected 55 , such targeted action could be advantageous in preserving motor function without exacerbating excitotoxicity or unwanted effect related to a global increase of neuronal activity. Finally, this study displays a trend towards an increased Ca 2+ influx -following TMZ treatment- into the hindbrain, a region controlling locomotor movements by reticulospinal neurons complex networks including Mauthner neurons which contact spinal motor neurons 56 , 57 . This suggests that TMZ could act also on higher centers involved in motor circuitry upstream of spinal cord. Once assessed the specific neuronal population involved, the elucidation of the detailed mechanism of action of TMZ in modulating motor neuron health and Ca 2+ fluxes -e.g. by acting on specific Ca 2+ channels or buffering systems- might be pursued. In conclusion, this study contributes clarifying the uncertain mechanism of action of this TMZ which, based on its preclinical efficacy, has recently grabbed the attention of the scientific community for the treatment of ALS (clinical trial NCT04788745), and further supports its reappraisal as a specific treatment to slow down the progression of motor symptoms in this disease. MATERIALS AND METHODS Zebrafish maintenance Experiments were carried out on a wild-type (WT) AB line and the transgenic [Tg( neurod1:GCaMP6f )] line (generously provided by Claire Wyart from the Institut du Cerveau et de la Moelle Épinière, Paris, France) 58 in the nacre ( mitfa −/− ) background. We had previously confirmed that there were no behavioral differences between the Tg( neurod1:GCaMP6f ) line and the standard AB line 59 . Adult zebrafish were kept in tanks with a density of no more than five fish per liter, at a constant temperature of 28°C, under a 14-hour light/10-hour dark cycle. Fertilized eggs and embryos were cultured at 28.5°C in egg water prepared with "Instant Ocean" sea salts (60 µg/mL) (Aquarium Systems, Sarrebourg, France) and in E3 medium (292.2 mg NaCl, 12.6 mg KCl, 48.6 mg CaCl 2 , and 39.8 mg MgSO 4 per 1 L of deionized water), following standard procedures 60 . The fish were staged by hours post fertilization (hpf) or days post fertilization (dpf). Locomotor behavior Larval locomotor behavior (distance traveled and velocity) of WT AB larvae at 4 days post fertilization (dpf) was assessed using the DanioVision system (Noldus Information Technology). Briefly, individual larvae were placed into 96-well plates with 300 µL of E3 medium per well. The plates were then inserted into the DanioVision system, where larval locomotor activity was recorded for 20 minutes. After this, TMZ was added to each well at increasing concentrations (25, 50, 100 and 200 µM), and larval locomotor activity was recorded again for 20 minutes. The recorded data were analyzed using EthoVision XT video-tracking software (Noldus Information Technology) to measure the distance traveled and the velocity of the larvae. Ca 2+ imaging recordings At 4 days post fertilization (dpf), zebrafish larvae were immobilized in low melting point agarose, with the larvae arranged caudal-laterally for spinal cord recordings and rostral-dorsally for whole brain and hindbrain recordings. A Nikon FN1 microscope (Nikon, Tokyo, Japan) was used for video recording, with image acquisitions made using a Prime sCMOS camera (Teledyne Photometrics, Tucson, AZ, USA) and Metafluor software (Molecular Devices, San Jose, CA). A time-lapse interval of 150 ms was applied, capturing 2412 frames per video. The recordings were conducted in two sessions: one before TMZ treatment and another after the treatment. A 1-minute wait period was observed between the two recording sessions to allow TMZ to diffuse through the sample. Fluorescence fluctuation distributions (DF/F0) were analyzed within a pre-defined region of interest (ROI) using ImageJ 64 software. Data were normalized to the background fluorescence and quantified by calculating the mean of the distribution. Statistics All data presented result from the analysis of three or more independent experiments. Statistics was conducted using GraphPad Prism 9. Distribution of the data was determined by the Shapiro-Wilk test and thus quantitative variables were analyzed using either parametric or non-parametric methods. For comparisons between two groups, the t-test was used for normally distributed data, while the Wilcoxon test was applied for non-normally distributed data. Statistical significance is indicated as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, or **** p ≤ 0.0001. The specific test used for each analysis is described in the figure legend. Declarations Ethical approval All animal procedures were conducted with the ethical approval of the Italian Ministry of Health (approval n° 584/2019-PR), in accordance with the European Union's Directive 2010/63/EU on the protection of animals used for scientific purposes, under the supervision of the University of Pisa Animal Care and Use Committee, and conformed to ARRIVE guidelines 2.0 to improve the reporting of research involving animals. The investigators declare that the principle of the “3R” (replace, reduce and refine) was carefully fulfilled. Data availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Author contributions E.F. and M.M. conceived this work and designed the experiments; S.B. and S.V. performed the experiments and analyzed the data; E.F., M.M., S.B., S.V. and C.G. carried out the interpretation of data; E.F. and M.M. supervised the study and wrote the manuscript text; E.F. and M.M. acquired funding and substantively revised the manuscript. All authors read, revised, and approved the final version of the manuscript. Funding sources This research was funded by the French Muscular Dystrophy Association AFM-TELETHON (Grant No. AFM 23771) to E.F., by the PRIN 2022 PNRR (Progetti di Ricerca di Rilevante Interesse Nazionale-National Recovery and Resilience Plan) funded by the European Union – NextGenerationEU–and adopted by the Italian Ministry of University and Research (Grant No. P2022LSW98) to E.F. and by the Telethon Foundation (grants GSA23C003 and GGP20011) to M.M. 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Delestrée, N., Manuel, M., Iglesias, C., Elbasiouny, S.M., Heckman, C.J., and Zytnicki, D. (2014). Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis. J Physiol 592 , 1687-1703. 10.1113/jphysiol.2013.265843. Martínez-Silva, M.L., Imhoff-Manuel, R.D., Sharma, A., Heckman, C.J., Shneider, N.A., Roselli, F., Zytnicki, D., and Manuel, M. (2018). Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS. Elife 7 . 10.7554/eLife.30955. Quinlan, K.A., Schuster, J.E., Fu, R., Siddique, T., and Heckman, C.J. (2011). Altered postnatal maturation of electrical properties in spinal motoneurons in a mouse model of amyotrophic lateral sclerosis. J Physiol 589 , 2245-2260. 10.1113/jphysiol.2010.200659. Leroy, F., Lamotte d'Incamps, B., Imhoff-Manuel, R.D., and Zytnicki, D. (2014). 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Increased persistent Na(+) current and its effect on excitability in motoneurones cultured from mutant SOD1 mice. J Physiol 563 , 843-854. 10.1113/jphysiol.2004.074138. Martin, E., Cazenave, W., Cattaert, D., and Branchereau, P. (2013). Embryonic alteration of motoneuronal morphology induces hyperexcitability in the mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 54 , 116-126. 10.1016/j.nbd.2013.02.011. Sareen, D., O'Rourke, J.G., Meera, P., Muhammad, A.K., Grant, S., Simpkinson, M., Bell, S., Carmona, S., Ornelas, L., Sahabian, A., et al. (2013). Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci Transl Med 5 , 208ra149. 10.1126/scitranslmed.3007529. Renton, A.E., Chiò, A., and Traynor, B.J. (2014). State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci 17 , 17-23. 10.1038/nn.3584. Naujock, M., Stanslowsky, N., Bufler, S., Naumann, M., Reinhardt, P., Sterneckert, J., Kefalakes, E., Kassebaum, C., Bursch, F., Lojewski, X., et al. (2016). 4-Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from Amyotrophic Lateral Sclerosis Patient-Derived Induced Pluripotent Stem Cells. Stem Cells 34 , 1563-1575. 10.1002/stem.2354. Sances, S., Bruijn, L.I., Chandran, S., Eggan, K., Ho, R., Klim, J.R., Livesey, M.R., Lowry, E., Macklis, J.D., Rushton, D., et al. (2016). Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nat Neurosci 19 , 542-553. 10.1038/nn.4273. Liu, Y., Pattamatta, A., Zu, T., Reid, T., Bardhi, O., Borchelt, D.R., Yachnis, A.T., and Ranum, L.P. (2016). C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD. Neuron 90 , 521-534. 10.1016/j.neuron.2016.04.005. Marchand-Pauvert, V., Peyre, I., Lackmy-Vallee, A., Querin, G., Bede, P., Lacomblez, L., Debs, R., and Pradat, P.F. (2019). Absence of hyperexcitability of spinal motoneurons in patients with amyotrophic lateral sclerosis. J Physiol 597 , 5445-5467. 10.1113/JP278117. Tremblay, E., Martineau, É., and Robitaille, R. (2017). Opposite Synaptic Alterations at the Neuromuscular Junction in an ALS Mouse Model: When Motor Units Matter. J Neurosci 37 , 8901-8918. 10.1523/JNEUROSCI.3090-16.2017. Saxena, S., Roselli, F., Singh, K., Leptien, K., Julien, J.P., Gros-Louis, F., and Caroni, P. (2013). Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival. Neuron 80 , 80-96. 10.1016/j.neuron.2013.07.027. Le Gall, L., Anakor, E., Connolly, O., Vijayakumar, U.G., Duddy, W.J., and Duguez, S. (2020). Molecular and Cellular Mechanisms Affected in ALS. J Pers Med 10 . 10.3390/jpm10030101. Hale, M.E., Katz, H.R., Peek, M.Y., and Fremont, R.T. (2016). Neural circuits that drive startle behavior, with a focus on the Mauthner cells and spiral fiber neurons of fishes. J Neurogenet 30 , 89-100. 10.1080/01677063.2016.1182526. Jontes, J.D., Buchanan, J., and Smith, S.J. (2000). Growth cone and dendrite dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo. Nat Neurosci 3 , 231-237. 10.1038/72936. Rupprecht, P., Prendergast, A., Wyart, C., and Friedrich, R.W. (2016). Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy. Biomed Opt Express 7 , 1656-1671. 10.1364/BOE.7.001656. Della Vecchia, S., Ogi, A., Licitra, R., Abramo, F., Nardi, G., Mero, S., Landi, S., Battini, R., Sicca, F., Ratto, G.M., et al. (2022). Trehalose Treatment in Zebrafish Model of Lafora Disease. Int J Mol Sci 23 . 10.3390/ijms23126874. Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene Additional Declarations No competing interests reported. Supplementary Files SupplementaryvideoS1.mp4 Supplementary video S1. Spinal cord Ca 2+ imaging Representative recordings of spinal cord neurons Ca 2+ imaging performed in the lateral caudal neurons of a single zebrafish larva before treatment (PRE-TMZ) and after treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ. SupplementaryvideoS2.mp4 Representative recordings of whole brain Ca 2+ imaging in the rostral-dorsally placed larvae before the treatment (PRE-TMZ) and after the treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ Supplementary video S2. Whole brain Ca 2+ imaging SupplementaryvideoS3.mp4 Supplementary video S3. Hindbrain Ca 2+ imaging Representative recordings of hindbrain Ca 2+ imaging performed in the larvae rostral-dorsally settled before the treatment (PRE-TMZ) and after the treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ. Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Apr, 2025 Reviews received at journal 31 Mar, 2025 Reviewers agreed at journal 06 Mar, 2025 Reviews received at journal 05 Feb, 2025 Reviewers agreed at journal 25 Jan, 2025 Reviewers agreed at journal 22 Jan, 2025 Reviewers invited by journal 18 Jan, 2025 Editor assigned by journal 18 Jan, 2025 Editor invited by journal 15 Jan, 2025 Submission checks completed at journal 15 Jan, 2025 First submitted to journal 10 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5803344","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":436092180,"identity":"27101e06-7c44-4688-882e-7ff9ef4776ee","order_by":0,"name":"Sara Bernardi","email":"","orcid":"","institution":"IRCCS Fondazione Stella Maris","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Bernardi","suffix":""},{"id":436092181,"identity":"66494c75-cb5f-42d1-a93f-4627c07a2b69","order_by":1,"name":"Sara Vitolo","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Vitolo","suffix":""},{"id":436092182,"identity":"c4ae0de5-2c19-45d0-84c2-4bfde53226f6","order_by":2,"name":"Chiara Gabellini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Gabellini","suffix":""},{"id":436092183,"identity":"04a61cb5-6abb-4f3b-91c1-06df93f6e6af","order_by":3,"name":"Maria Marchese","email":"","orcid":"","institution":"IRCCS Fondazione Stella Maris","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Marchese","suffix":""},{"id":436092185,"identity":"d73aa387-73a1-4ad4-9b4d-c31e3ee32fb2","order_by":4,"name":"Elisabetta Ferraro","email":"data:image/png;base64,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","orcid":"","institution":"University of Pisa","correspondingAuthor":true,"prefix":"","firstName":"Elisabetta","middleName":"","lastName":"Ferraro","suffix":""}],"badges":[],"createdAt":"2025-01-10 11:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5803344/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5803344/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-06065-y","type":"published","date":"2025-07-02T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79787874,"identity":"8e648061-e4ab-4958-bb94-7bf4ee365fa3","added_by":"auto","created_at":"2025-04-02 17:17:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1238553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocomotor behavior in TMZ-treated zebrafish larvae.\u003c/strong\u003e (A) Distance traveled (mm) and (B) velocity (mm/s) of zebrafish larvae pre- and post-TMZ administration at increasing TMZ concentrations. (C) Representative movement track pre- and post-TMZ (200μM) administration. n range=34-84 larvae/group; specifically indicated in each panel. Statistical analysis was performed using the Wilcoxon test- Asterisks denote significance; **** p ≤ 0.0001). Data are presented as Mean±SEM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/c811cfd5a6a1fe6c4a86efbf.png"},{"id":79787875,"identity":"cbf40cd2-553e-4111-81ac-3b3eaa6ab326","added_by":"auto","created_at":"2025-04-02 17:17:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4517293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpinal cord Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eimaging upon TMZ exposure. (A) \u003c/strong\u003eCa\u003csup\u003e2+\u003c/sup\u003e imaging in spinal cord neurons of Tg(\u003cem\u003eneurod1\u003c/em\u003e:GCaMP6f) larvae was performed 10 minutes (min) before 100 µM TMZ treatment for baseline recordings (PRE-TMZ). After TMZ administration and after a TMZ-diffusion time of 1 min, calcium imaging was resumed for other 10 min (POST-TMZ). \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative z-projection images of the spinal cord Ca\u003csup\u003e2+\u003c/sup\u003e imaging performed in the lateral caudal neurons of a single zebrafish larva before the treatment (PRE-TMZ) and after the treatment (POST-TMZ), the Region of Interest (ROI) is highlighted with the white dotted line). On the right side, the look up table color range of Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence is shown. \u003cstrong\u003e(C\u003c/strong\u003e) A representative graph from a single zebrafish larva shows the ∆F/F0 calcium fluorescent signal in spinal cord for each frame\u003cstrong\u003e \u003c/strong\u003ebefore and after TMZ treatment \u003cstrong\u003e(D\u003c/strong\u003e) The graph shows the full distribution of data with the median and quartiles of ∆F/F0 fluorescent signal in spinal neurons. n=10 analyzed larvae\u003cstrong\u003e. \u003c/strong\u003eStatistical analysis was performed using the Wilcoxon test. Asterisks denote significance; **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/65aa322c926b8e918d94f4da.png"},{"id":79787876,"identity":"2f2edfce-8db8-4a53-acb3-a1b01acc2612","added_by":"auto","created_at":"2025-04-02 17:17:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3769031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole brain \u003c/strong\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003cstrong\u003e imaging upon TMZ exposure. (A) \u003c/strong\u003eRepresentative z-projection images of the whole brain Ca\u003csup\u003e2+\u003c/sup\u003e imaging performed in the rostral-dorsally placed Tg(\u003cem\u003eneurod1\u003c/em\u003e:GCaMP6f) larvae before the treatment (PRE-TMZ) and after the treatment (POST-TMZ). On the right side, the look up table color range of Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence is shown. Images were taken with 4X magnification objective. The dotted line indicates the analyzed ROI.\u003cstrong\u003e (B\u003c/strong\u003e) A representative graph from a single zebrafish larva shows the ∆F/F0 Ca\u003csup\u003e2+\u003c/sup\u003e fluorescent signal in the whole brain for each frame\u003cstrong\u003e \u003c/strong\u003ebefore and after TMZ treatment \u003cstrong\u003e(C\u003c/strong\u003e) The graph shows full distribution of data, with median and quartiles of ∆F/F0 fluorescent signal in whole brain\u003cstrong\u003e.\u003c/strong\u003e n=10 analyzed larvae.\u003cstrong\u003e \u003c/strong\u003eStatistical analysis was performed using the Wilcoxon test. ns: not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/27da008d72d6494923f16a15.png"},{"id":79788274,"identity":"ec92a3c3-bade-46db-bcb5-26537d32faac","added_by":"auto","created_at":"2025-04-02 17:25:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5288642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHindbrain \u003c/strong\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003cstrong\u003e imaging upon TMZ exposure. (A) \u003c/strong\u003eRepresentative z-projection images of the hindbrain calcium imaging performed in Tg(\u003cem\u003eneurod1\u003c/em\u003e:GCaMP6f)\u0026nbsp; larvae rostral-dorsally settled before the treatment (PRE-TMZ) and after the treatment (POST-TMZ). On the right side, the look up table color range of Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence is shown. The dotted line indicates the analyzed ROI, while the white arrows indicate the Mauthner cells localization. \u003cstrong\u003e(B\u003c/strong\u003e) A representative graph from a single zebrafish larva shows the ∆F/F0 Ca\u003csup\u003e2+\u003c/sup\u003e fluorescent signal in hindbrain for each frame\u003cstrong\u003e \u003c/strong\u003ebefore and after TMZ treatment. \u003cstrong\u003e(C\u003c/strong\u003e) The graph shows full distribution of data, with median and quartiles of ∆F/F0 fluorescent signal in in the hindbrain\u003cstrong\u003e.\u003c/strong\u003e n=10 analyzed larvae.\u003cstrong\u003e \u003c/strong\u003eStatistical analysis was performed using the Wilcoxon test. ns: not significant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/3422abeb33a40806a0dcc549.png"},{"id":86179198,"identity":"ca74d4ee-100b-49e7-ab18-6efdb0d89f35","added_by":"auto","created_at":"2025-07-07 16:17:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13767516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/bc378658-c20f-4c1f-b847-15ef48fa4d53.pdf"},{"id":79788279,"identity":"a0863072-943d-4e4a-a36c-41a54b2c230e","added_by":"auto","created_at":"2025-04-02 17:25:28","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12869291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary video S1. Spinal cord Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eimaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative recordings of spinal cord neurons Ca\u003csup\u003e2+\u003c/sup\u003e imaging performed in the lateral caudal neurons of a single zebrafish larva before treatment (PRE-TMZ) and after treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ.\u003c/p\u003e","description":"","filename":"SupplementaryvideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/3aa0cea7f2f8aa8635107044.mp4"},{"id":79788280,"identity":"7fd41eb5-d5f4-4df4-a5e4-7a66a828bb7e","added_by":"auto","created_at":"2025-04-02 17:25:28","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12727841,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative recordings of whole brain Ca\u003csup\u003e2+\u003c/sup\u003e imaging in the rostral-dorsally placed larvae before the treatment (PRE-TMZ) and after the treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary video S2. Whole brain Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e imaging\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryvideoS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/98cb063b0cf473cc1cf74470.mp4"},{"id":79787884,"identity":"e4423717-ef0b-4d62-b2e7-c06e20dc4718","added_by":"auto","created_at":"2025-04-02 17:17:27","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4094467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary video S3. Hindbrain Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative recordings of hindbrain Ca\u003csup\u003e2+\u003c/sup\u003e imaging performed in the larvae rostral-dorsally settled before the treatment (PRE-TMZ) and after the treatment (POST-TMZ) (100 µM), recordings were speed up at 70 fps with ImageJ.\u003c/p\u003e","description":"","filename":"SupplementaryvideoS3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5803344/v1/b43db5bbeedbf2f4e846ab21.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTrimetazidine, a promising drug for amyotrophic lateral sclerosis, modulates Ca\u003csup\u003e2+\u003c/sup\u003e influx in spinal neurons\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTrimetazidine (TMZ) is a antianginal beneficial on ischemic injury \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Also, it has more recently been demonstrated that TMZ acts on skeletal muscle by promoting myogenesis, by improving muscle quality and neuromuscular communication, and by increasing muscle strength in mice models of aging and cachexia, as well as in patients with peripheral arterial disease \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, we have reported that orally administered TMZ leads to motor performance improvement in transgenic SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, the standard animal model for Amyotrophic Lateral Sclerosis (ALS) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. TMZ is a piperazine derivative inhibiting the 3-ketoacyl coenzyme A thiolase which catalyzes long-chain fatty acid β-oxidation, thus leading to a shift from fatty acid to glucose as preferential used substrate. This metabolic shift has been found to optimize energy production \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, evidence for this mechanism is controversial and since, based on its preclinical efficacy, TMZ is under clinical trial (NCT04788745), elucidating the details of its action is a high-priority.\u003c/p\u003e \u003cp\u003eALS is a neurodegenerative disease affecting both upper corticospinal motor neurons and lower ones in brainstem and spinal cord. ALS progresses from weakness to gradual muscle atrophy and inability to perform movements, till complete paralysis and death \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Etiopathogenesis of ALS is poorly understood and, also for this reason, it currently lacks effective treatment. Also prior to clinical symptoms, electively vulnerable motor neurons, responsible for voluntary muscle contraction, show early pathological signs including axonal transport defects, protein aggregation, mitochondrial dysfunctions and altered intrinsic excitability \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. It has been proposed that motor neuron electrical signaling initially increases during the early stages of ALS and then it gradually decreases, as neurons degenerate, and lose their ability to properly respond to stimuli \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Notably, both neuronal hyperexcitability and hypoexcitability have been observed in ALS; however, it is not clear if they are compensatory or pathological events \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Also, disassembly and denervation of the neuromuscular junction (NMJ) occur in ALS before motor neuron degeneration, with episodes of reinnervation interpreted as attempts to regenerate NMJ \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSince among the mechanisms proposed to explain the origin of ALS are mitochondrial and metabolic dysfunctions \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, we have recently focused our attention on evaluating and proving the pre-clinical efficacy of the metabolic modulator TMZ in counteracting ALS progression \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, its mechanism is controversial and, in addition, we had previously observed that murine skeletal muscles treated with TMZ \u003cem\u003eex vivo\u003c/em\u003e display a very quick shift towards a slow-twitch contractile phenotype \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This very rapid effect might not be explained by gene expression modulation therefore we hypothesized an ability of this drug to modify ionic fluxes. To address this issue, we focused our attention on Ca\u003csup\u003e2+\u003c/sup\u003e since it is the final ionic effector of both skeletal muscle contraction and motor neuron firing. This choice was also supported by previous studies demonstrating the ability - not clearly elucidated- of TMZ to act on Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis in cardiomyocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo study the role of TMZ on Ca\u003csup\u003e2+\u003c/sup\u003e dynamics in neurons and neuronal activity \u003cem\u003ein vivo\u003c/em\u003e, we took advantage of a model represented by the transgenic zebrafish line \u003cem\u003eTg(neurod1\u003c/em\u003e:GCaMP6f\u003cem\u003e)\u003c/em\u003e for \u003cem\u003ein vivo\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e imaging in neurons \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. By this tool we visualized Ca\u003csup\u003e2+\u003c/sup\u003e dynamics in neuronal cells demonstrating the ability of TMZ to induce Ca\u003csup\u003e2+\u003c/sup\u003e influx and spinal neuron firing which correlates with zebrafish motor performance stimulation by this drug.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eTMZ improves locomotor performance in zebrafish larvae\u003c/h2\u003e\n \u003cp\u003eIn order to confirm the effect of TMZ on locomotion also on zebrafish larvae, which we used as model to evaluate Ca\u003csup\u003e2+\u003c/sup\u003e fluxes, we first performed a toxicity assay using increasing concentrations of TMZ, specifically 25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, 200 \u0026micro;M, 1000 \u0026micro;M and 2000 \u0026micro;M. We administered the drug to 4 dpf larvae and we kept them in these solutions for 24h. We observed that 1000 \u0026micro;M and 2000 \u0026micro;M TMZ were toxic for the larvae which died a few hours upon drug administration. For this reason, we performed the behavior locomotor assay by administrating 25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M and 200 \u0026micro;M TMZ to 4 dpf larvae to obtain the velocity and the distance travelled by the larvae pre- and post-TMZ treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Our data confirm that there is a statistically significant improvement of the locomotor performance upon both 100 \u0026micro;M and 200 \u0026micro;M TMZ treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). These data are in line with the results obtained by treating the Tg SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model of ALS and that of aging with TMZ which increased their locomotory activity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and make zebrafish larvae a suitable model to unravel the mechanisms underlying TMZ effect on locomotion.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCa\u003csup\u003e2+\u003c/sup\u003e influx in spinal cord is stimulated by TMZ\u003c/h3\u003e\n\u003cp\u003eBased on the analysis of zebrafish locomotor behaviour upon TMZ treatment, along with the previous observation that \u003cem\u003eex vivo\u003c/em\u003e administration of 100 \u0026micro;M TMZ is able to act very quickly on the contraction rate of excised \u003cem\u003etibialis anterior\u003c/em\u003e murine muscles \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, we hypothesized that this metabolic modulator might be able to act on skeletal muscle contractility and, consequently, on locomotion, by modifying ionic fluxes. Therefore, we decided to evaluate the firing of the skeletal muscle-stimulating motor neurons through the analysis of Ca\u003csup\u003e2+\u003c/sup\u003e fluxes \u003cem\u003ein vivo\u003c/em\u003e. We used the transgenic zebrafish line \u003cem\u003eTg(neurod1\u003c/em\u003e:GCaMP6f\u003cem\u003e)\u003c/em\u003e based on the \u003cem\u003eneurod1\u003c/em\u003e promoter-mediated neuronal expression of the GFP-based Ca\u003csup\u003e2+\u003c/sup\u003e indicator GCaMP6f .\u003c/p\u003e\n\u003cp\u003e100 \u0026micro;M TMZ was administered to 4 dpf Tg(\u003cem\u003eneurod1\u003c/em\u003e:GCaMP6f) larvae and Ca\u003csup\u003e2+\u003c/sup\u003e imaging was first performed on spinal cord (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; 40X magnification). The images were acquired before and after TMZ treatment with the timing described in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. By comparing the fluorescence signal derived from GCaMP activation before and after TMZ stimulation, a significant increase in Ca\u003csup\u003e2+\u003c/sup\u003e influx- and thus neuron firing- was observed upon TMZ administration (see the representative images in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and video recordings in Supplementary Video S1). Accordingly, the ∆F/F0 ratio over time shows a higher fluorescent signal recorded following TMZ treatment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC; POST-TMZ) compared to the signal obtained from the untreated larvae (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC; PRE-TMZ). The statistical analyses confirms that TMZ was effective in increasing Ca\u003csup\u003e2+\u003c/sup\u003e influx in neurons of the spinal cord where motor neurons are localized (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Also spinal interneurons modulating motor neuron activity are highlighted in this Tg model, therefore, by this experiment it is not possible to precisely identify the neuronal population targeted by TMZ which might likely be represented by motor neurons (indeed is it possible to observe the axon leaving the spinal cord, which indicates that motor neurons are involved; see Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplementary video S1) or by interneurons modulating motor neuron activity.\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cstrong\u003eTMZ does not induce an overall brain increase of Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003einflux\u003c/strong\u003e\u003c/div\u003e\n\u003cp\u003eIn order to evaluate if the effect of TMZ was specific to the spinal cord or was occurring in all CNS neurons, we visualized whole brain activity by recording Ca\u003csup\u003e2+\u003c/sup\u003e imaging at lower magnification (4X). The images were acquired before and after TMZ treatment with the timing described in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. Differently from what we had previously observed in the spinal cord (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), no increase in the whole brain Ca\u003csup\u003e2+\u003c/sup\u003e influx was observed after TMZ treatment; see representative images (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), videos (Supplementary video S2) and graph (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eHowever, we decided to focus more specifically on the hindbrain region where neurons contacting and modulating motor neurons are localized, in order to get information on the kind of neurons targeted by TMZ which might be directly targeted motor neurons or spinal interneurons or also higher center neurons controlling locomotor movements. For this reason, the same magnification used for spinal cord analysis (40X) was used for the hindbrain recordings. The region chosen for the analysis is an area surrounding the Mauthner cells within the hindbrain (defined by a dotted line in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). This analysis revealed an increase of the fluorescence signal in the hindbrain region of Tg(\u003cem\u003eneurod1\u003c/em\u003e:GCaMP6f) larvae upon TMZ administration (see the Supplementary video S3 and the representative \u0026Delta;F/F0 graph in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), which is, however, not statistically significant (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Nevertheless, although not reaching the significance, a relevant trend to an increase of Ca\u003csup\u003e2+\u003c/sup\u003e influx following TMZ treatment was recorded (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Considering that the Mauthner cells region is involved in fish escape response, this suggests that TMZ might also act on hindbrain neurons involved in the upstream motor circuitry controlling motor neurons.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study presents, to our knowledge, the first compelling evidence that TMZ significantly enhances Ca\u0026sup2;⁺ influx in spinal neurons, indicating a potential role for this drug in modulating Ca\u0026sup2;⁺ dynamics. This modulation may help explain the enhanced locomotion parameters induced by TMZ on aging and ALS mouse models \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and confirmed in this study on zebrafish. In the context of ALS, where motor neuron degeneration and progressive loss of muscle function occur \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, TMZ's ability to improve locomotion is noteworthy. However, it is well-documented that elevated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels contribute to neurotoxicity, and that altered Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis is a pathological feature of ALS \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. On the other hand, even though hyperexcitability and fasciculations are features of ALS \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and therefore increased excitability triggered by TMZ might, at first glance, be considered detrimental, some issues need to be considered.\u003c/p\u003e \u003cp\u003eFirst of all, hyperexcitability and fasciculations origin and contribution to the pathophysiology of ALS are unknown. It has been proposed that proximal and distal factors exist; intrinsic excitability of motor neurons, supraspinal excitability from upper motor neurons and altered regulation by interneurons, Renshaw cells or propriospinal neurons -possibly associated to excitotoxic synaptic milieu likely due to altered glutamate levels. Also, it has been suggested that, with the progressive dysfunction of lower motor neurons, fasciculations arise in abnormal, reinnervated motor units; then, as motor neurons degenerate, fasciculations become less prominent. Fasciculations might therefore occur along with compensatory -but insufficient- reinnervation, thus possible being early symptoms of aberrant firing of motor neurons \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and, therefore, hyperexcitability might not necessarily be the cause of neurodegeneration. In this scenario, it can be hypothesized that drugs triggering Ca\u003csup\u003e2+\u003c/sup\u003e influx, spinal neuron excitability and consequent skeletal muscle contraction might be compensatory and aiming at counteracting neurodegeneration and avoiding NMJ denervation. This is in accordance with our previous finding that TMZ protects against NMJ impairment \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother important issue worth to be discussed concerns the occurrence of hypoexcitability in spinal motoneurons of ALS mouse models \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, whereas cortical hyperexcitability has been described as an early feature in ALS thought to contribute to neuronal stress and subsequent degeneration \u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been reported that motor neurons derived from induced pluripotent stem cells obtained from ALS patients exhibit hyperexcitability and/or hypoexcitability \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan additionalcitationids=\"CR48 CR49 CR50\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and that motor neurons recorded from ALS patients are hypoexcitable \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Based on some author\u0026rsquo;s hypothesis, motor neurons do not develop hyperexcitability before NMJ denervation, but some motor neurons fails to produce sustained firing, and hypoexcitability occurs, despite the fact that their NMJs are still functional \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It has been proposed that hypoexcitability might lead to peripheral disconnection and to reduced functionality of fast fatigable NMJ and that hyperexcitability might arise in the remaining resistant neurons and compensate for the hypoexcitable vulnerable ones \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In this view, hypoexcitability might be the earliest pathological sign of some motor neurons -possibly due to the accumulation of misfolded SOD1 proteins- leading to dysfunctional motor units, whereas hyperexcitability might be neuroprotective and compensatory \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo sum up, despite the discussed hypothesis, it is still obscure whether hyperexcitability and hypoexcitability are compensatory or represent pathological traits in ASL. Therefore, we believe that the potential beneficial therapeutic action of TMZ, a drug transiently enhancing motor neuron excitability, protecting against the dismantlement of NMJs and improving locomotory abilities \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e deserves consideration and a thorough investigation. Further investigation is needed to identify the specific spinal neuronal population targeted by TMZ; indeed, we cannot distinguish motor neuron somata from large spinal interneurons modulating motor neuron activity, even though the observation of the axon leaving the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Supplementary video S1) indicate that motor neurons are involved. To elucidate this issue, transgenic zebrafish lines for \u003cem\u003ein vivo\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e imaging in specific spinal neuron populations would be helpful.\u003c/p\u003e \u003cp\u003eThese data also reveal an interesting specificity of TMZ effect, which selectively affects spinal neurons, thus suggesting a particular effectiveness in modulating motor control. In ALS, where the specific motor neuronal population is affected \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, such targeted action could be advantageous in preserving motor function without exacerbating excitotoxicity or unwanted effect related to a global increase of neuronal activity. Finally, this study displays a trend towards an increased Ca\u003csup\u003e2+\u003c/sup\u003e influx -following TMZ treatment- into the hindbrain, a region controlling locomotor movements by reticulospinal neurons complex networks including Mauthner neurons which contact spinal motor neurons \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This suggests that TMZ could act also on higher centers involved in motor circuitry upstream of spinal cord. Once assessed the specific neuronal population involved, the elucidation of the detailed mechanism of action of TMZ in modulating motor neuron health and Ca\u003csup\u003e2+\u003c/sup\u003e fluxes -e.g. by acting on specific Ca\u003csup\u003e2+\u003c/sup\u003e channels or buffering systems- might be pursued.\u003c/p\u003e \u003cp\u003eIn conclusion, this study contributes clarifying the uncertain mechanism of action of this TMZ which, based on its preclinical efficacy, has recently grabbed the attention of the scientific community for the treatment of ALS (clinical trial NCT04788745), and further supports its reappraisal as a specific treatment to slow down the progression of motor symptoms in this disease.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eZebrafish maintenance\u003c/h2\u003e \u003cp\u003eExperiments were carried out on a wild-type (WT) AB line and the transgenic [Tg(\u003cem\u003eneurod1:GCaMP6f\u003c/em\u003e)] line (generously provided by Claire Wyart from the Institut du Cerveau et de la Moelle \u0026Eacute;pini\u0026egrave;re, Paris, France) \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e in the nacre (\u003cem\u003emitfa\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) background. We had previously confirmed that there were no behavioral differences between the Tg(\u003cem\u003eneurod1:GCaMP6f\u003c/em\u003e) line and the standard AB line \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Adult zebrafish were kept in tanks with a density of no more than five fish per liter, at a constant temperature of 28\u0026deg;C, under a 14-hour light/10-hour dark cycle. Fertilized eggs and embryos were cultured at 28.5\u0026deg;C in egg water prepared with \"Instant Ocean\" sea salts (60 \u0026micro;g/mL) (Aquarium Systems, Sarrebourg, France) and in E3 medium (292.2 mg NaCl, 12.6 mg KCl, 48.6 mg CaCl\u003csub\u003e2\u003c/sub\u003e, and 39.8 mg MgSO\u003csub\u003e4\u003c/sub\u003e per 1 L of deionized water), following standard procedures \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The fish were staged by hours post fertilization (hpf) or days post fertilization (dpf).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLocomotor behavior\u003c/h3\u003e\n\u003cp\u003eLarval locomotor behavior (distance traveled and velocity) of WT AB larvae at 4 days post fertilization (dpf) was assessed using the DanioVision system (Noldus Information Technology). Briefly, individual larvae were placed into 96-well plates with 300 \u0026micro;L of E3 medium per well. The plates were then inserted into the DanioVision system, where larval locomotor activity was recorded for 20 minutes. After this, TMZ was added to each well at increasing concentrations (25, 50, 100 and 200 \u0026micro;M), and larval locomotor activity was recorded again for 20 minutes. The recorded data were analyzed using EthoVision XT video-tracking software (Noldus Information Technology) to measure the distance traveled and the velocity of the larvae.\u003c/p\u003e\n\u003ch3\u003eCa\u003csup\u003e2+\u003c/sup\u003e imaging recordings\u003c/h3\u003e\n\u003cp\u003eAt 4 days post fertilization (dpf), zebrafish larvae were immobilized in low melting point agarose, with the larvae arranged caudal-laterally for spinal cord recordings and rostral-dorsally for whole brain and hindbrain recordings. A Nikon FN1 microscope (Nikon, Tokyo, Japan) was used for video recording, with image acquisitions made using a Prime sCMOS camera (Teledyne Photometrics, Tucson, AZ, USA) and Metafluor software (Molecular Devices, San Jose, CA). A time-lapse interval of 150 ms was applied, capturing 2412 frames per video. The recordings were conducted in two sessions: one before TMZ treatment and another after the treatment. A 1-minute wait period was observed between the two recording sessions to allow TMZ to diffuse through the sample. Fluorescence fluctuation distributions (DF/F0) were analyzed within a pre-defined region of interest (ROI) using ImageJ 64 software. Data were normalized to the background fluorescence and quantified by calculating the mean of the distribution.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll data presented result from the analysis of three or more independent experiments. Statistics was conducted using GraphPad Prism 9. Distribution of the data was determined by the Shapiro-Wilk test and thus quantitative variables were analyzed using either parametric or non-parametric methods. For comparisons between two groups, the t-test was used for normally distributed data, while the Wilcoxon test was applied for non-normally distributed data. Statistical significance is indicated as follows: * p\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, or **** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001. The specific test used for each analysis is described in the figure legend.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were conducted with the ethical approval of the Italian Ministry of Health (approval n\u0026deg; 584/2019-PR), in accordance with the European Union\u0026apos;s Directive 2010/63/EU on the protection of animals used for scientific purposes, under the supervision of the University of Pisa Animal Care and Use Committee, and conformed to ARRIVE guidelines 2.0 to improve the reporting of research involving animals. The investigators declare that the principle of the \u0026ldquo;3R\u0026rdquo; (replace, reduce and refine) was carefully fulfilled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, upon reasonable request.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.F. and M.M. conceived this work and designed the experiments; S.B. and S.V. performed the experiments and analyzed the data; E.F., M.M., S.B., S.V. and C.G. carried out the interpretation of data; E.F. and M.M. supervised the study and wrote the manuscript text; E.F. and M.M. acquired funding and substantively revised the manuscript. All authors read, revised, and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was funded by the French Muscular Dystrophy Association AFM-TELETHON (Grant No. AFM 23771) to E.F., by\u0026nbsp;the PRIN 2022 PNRR (Progetti di Ricerca di Rilevante Interesse Nazionale-National Recovery and Resilience Plan) funded by the European Union \u0026ndash; NextGenerationEU\u0026ndash;and adopted by the Italian Ministry of University and Research (Grant No. P2022LSW98) to E.F. and by the Telethon Foundation (grants GSA23C003 and GGP20011) to M.M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMonti, L.D., Setola, E., Fragasso, G., Camisasca, R.P., Lucotti, P., Galluccio, E., Origgi, A., Margonato, A., and Piatti, P. 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A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amyotrophic lateral sclerosis, Trimetazidine, Aging, Spinal cord, Skeletal muscle, Motor neuron disease, NMJ","lastPublishedDoi":"10.21203/rs.3.rs-5803344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5803344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe metabolic modulator trimetazidine (TMZ) is an antianginal recently found to improve skeletal muscle performance in mice models of sarcopenia and Amyotrophic Lateral Sclerosis (ALS). The mechanism underlying the effect of TMZ on locomotor activity has been proposed to rely on its ability to enhance metabolic efficiency with a consequent improvement of myogenesis and of neuromuscular junction (NMJ) and muscle function. However, although promising and therefore under clinical trials, the mechanism of action of TMZ has not been clearly disclosed; here we hypothesized that it might involve the modulation of neuronal Ca\u003csup\u003e2+\u003c/sup\u003e flows. We studied the effect of TMZ on Ca\u003csup\u003e2+\u003c/sup\u003e dynamics \u003cem\u003ein vivo\u003c/em\u003e, by using the transgenic zebrafish line \u003cem\u003eTg(neurod1\u003c/em\u003e:GCaMP6f\u003cem\u003e)\u003c/em\u003e in which the neuronal expression of the Ca\u003csup\u003e2+\u003c/sup\u003e indicator GCaMP allows to visualize Ca\u003csup\u003e2+\u003c/sup\u003e dynamics in neurons of zebrafish larvae. By this elegant tool, we demonstrated, for the first time, that TMZ promotes Ca\u003csup\u003e2+\u003c/sup\u003e influx in zebrafish spinal neurons likely enhancing motor neuron firing, which correlates with enhanced motor performance by this drug. Even though elevated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels have often been associated to neurotoxicity, it is unclear if the neuronal excitability features in ALS are compensatory or pathological. Therefore, TMZ might potentially contribute to counteract neurodegeneration by modulating neuronal Ca\u003csup\u003e2+\u003c/sup\u003e fluxes, and transiently and selectively enhancing motor neuron firing, as well as NMJ and locomotor function, without increasing the overall neuronal excitability. This further supports TMZ repurposing for the treatment of ALS and of other conditions characterized by NMJ impairment, such as aging.\u003c/p\u003e","manuscriptTitle":"Trimetazidine, a promising drug for amyotrophic lateral sclerosis, modulates Ca2+ influx in spinal neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 17:17:22","doi":"10.21203/rs.3.rs-5803344/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-03T06:03:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T04:19:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311612212421849688110195184990920800028","date":"2025-03-06T15:12:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-05T09:30:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307217800418674327863074106554218839077","date":"2025-01-25T19:48:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88897764155425563723986829148771350399","date":"2025-01-22T08:53:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-18T15:35:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-18T15:33:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-15T09:45:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-15T06:48:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-10T11:25:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe29499e-d7a9-413b-99f9-d7c5febddec0","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46593854,"name":"Health sciences/Neurology/Neurological disorders/Motor neuron disease/Amyotrophic lateral sclerosis"},{"id":46593855,"name":"Biological sciences/Neuroscience/Motor control/Neuromuscular junction"},{"id":46593856,"name":"Biological sciences/Neuroscience/Motor control/Spinal cord"},{"id":46593857,"name":"Biological sciences/Physiology/Ageing"},{"id":46593858,"name":"Biological sciences/Drug discovery/Target identification"}],"tags":[],"updatedAt":"2025-07-07T16:07:30+00:00","versionOfRecord":{"articleIdentity":"rs-5803344","link":"https://doi.org/10.1038/s41598-025-06065-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:58:29","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-04-02 17:17:22","video":"","vorDoi":"10.1038/s41598-025-06065-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-06065-y","workflowStages":[]},"version":"v1","identity":"rs-5803344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5803344","identity":"rs-5803344","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}