Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that influences motor neurons in the brain, brainstem, and spinal cord. Common symptoms of ALS include weakness, weight loss, and muscle atrophy, which ultimately lead to muscle paralysis (1-3). To date, mutations in more than 50 genes have been associated with ALS pathogenesis. Among these, fused in sarcoma (
Acid sphingomyelinase (ASM) encoded by
Herein, our study showed an increased ASM activity in the plasma or spinal cord of ALS patients and in a mouse model. Moreover, we found that genetic inhibition of ASM improved motor behavioral dysfunction and motor neuronal loss in the spinal cord of an ALS mouse model. Thus, our study provided evidence that increased ASM may influence ALS pathology, suggesting the possibility of ASM as a therapeutic target for ALS.
We first confirmed whether ASM activity was altered in plasma samples of ALS patients with
Next, to assess the potential effect of genetic ASM inhibition on motor function in FUS-R521C mice, we performed the tail suspension, hanging wire, and rotarod tests. Fig. 2A shows that FUS-R521C mice exhibited growth retardation, spastic paraplegia, and severe muscle wasting, but not in FUS-R521C;
Motor neuron loss in the spinal cord is generally observed in ALS pathologies, and the FUS-R521C mutant is associated with motor neuron degeneration (23, 24). Based on this, we confirmed motor neuron survival using an antibody against choline acetyl transferase (ChAT) to visualize and quantitate spinal motor neurons. The number of ChAT+ motor neurons was significantly reduced in the spinal cord of the FUS-R521C mice. However, the FUS-R521C;
Although the causative pathogenic mechanisms of ALS remain unclear, the complex interactions between genetic and environmental factors contribute to the development and progression of the disease. ASM is increased by environmental stress and several neurodegenerative diseases (14). Previous studies have shown that patients with ALS and mouse models have high levels of ceramide in the spinal cord, which affects inflammation, the loss of neuromuscular junctions, and the degeneration of motor neurons (26). In this study, we revealed that ASM activity increased in samples from ALS patients and FUS-R521C mice, indicating that increased ASM correlated with high levels of ceramide in ALS. Moreover, this finding suggests that elevation of ASM activity could influence the pathogenesis of ALS through ceramide.
Genetic inhibition of ASM improved motor behavioral function and spinal neuronal loss in FUS-R521C mice without affecting the expression of FUS-R521C proteins in the nucleus and cytoplasm. Mutations in
The most widely used drugs for ALS are those that manage symptoms, prevent complications, and slow down progression. However, since the mechanisms of action are likely to be varied and complex, they have failed to show significant efficacy in ALS (6, 7). Another treatment strategy is RNA-targeting therapeutics, such as short interfering RNA (siRNA). siRNA is used to decrease the expression of target genes by interacting with the RNA-induced silencing complex. This treatment has also been delayed by inefficient and poorly targeted delivery (6, 7). Small molecules have been investigated as alternatives and potential therapeutics for ALS. These small molecules are compounds with anti-inflammatory, anti-oxidative, anti-apoptotic, autophagy-inducing, and neuroprotective properties (29). Although the vast majority of compounds have limitations in the exact mode of action and their efficacy, the concomitant use of some compounds is allowed for the treatment of ALS.
Previous studies have suggested that several antidepressant drugs affect ASM inhibition through action as functional inhibitors (17, 30). However, these inhibitors lack specificity, have off-target effects, and have unclear mechanisms of therapeutic action in neurodegenerative diseases. Based on this, a new small compound was recently identified as a selective and direct ASM inhibitor without off-target effects (31). This ASM inhibitor is significantly effective in improving neuroinflammation, autophagy dysfunction, synapse loss, neuronal survival, and activity in neurodegenerative diseases (31). Moreover, this inhibitor exhibits excellent bioavailability, central nervous system distribution, and microsomal stability. Although ASM activity and its inhibitory efficacy should be further investigated in various ALS mouse models, this ASM inhibitor could be a potential therapeutic agent for ALS. Furthermore, we believe that the combination of ASM inhibitors with current therapeutic agents might present synergetic effects for the improvement of ALS pathology.
The following mouse lines were used: C57BL/6 WT mice (The Jackson Laboratory), FUS-R521C transgenic mice (22) (C57BL/6 background), and
Blood was collected into sodium heparin-coated tubes via intracardial bleeding at the time of death. Plasma was generated by centrifugation of freshly collected blood, and aliquots were stored at −80°C until use. Human plasma samples were obtained from individuals with ALS and age-matched controls from Hanyang University Hospital. Informed consent was obtained from all subjects according to the ethics committee guidelines of Hanyang University Hospital.
Enzymatic activity was measured as previously described (15, 31) using a UPLC system (Waters). Briefly, the spinal cord was lysed in a homogenization buffer containing 50 mM HEPES (Sigma-Aldrich, H3375), 150 mM NaCl (Sigma-Aldrich, S3014), 0.2% Igepal CA-630 (Sigma-Aldrich, I8896), and protease inhibitors (Calbiochem, 539131). Three microliters of the samples (plasma or spinal cord) were mixed with 3 μl of 200 μm BODIPY-C12-sphingomyelin (Invitrogen, D7711) diluted in 0.2 M of sodium acetate buffer, pH 5.0, 0.2 mM ZnCl2, and 0.2% Igepal CA-630, and incubated at 37°C for 1 h. Hydrolysis reactions were stopped by adding 114 μl and centrifuged at 13,000 rpm for 5 min. Thirty microliters of the supernatant was then transferred to a sampling glass vial, and 5 μl was applied to a UPLC system for analysis. Quantification was achieved by comparison with BODIPY-C12-ceramide using the Waters Millennium software.
For immunofluorescence staining, the spinal cord was cut using a vibratome (30 μm). ChAT antibody (1:100, Millipore, AB144p), FLAG-M2 (1:100, Sigma-Aldrich, F3165), and FUS (1:100, Sigma-Aldrich, HPA008784) were used to stain the neurons and FUS proteins in the spinal cord. Sections were analyzed using a laser-scanning confocal microscope (FV3000; Olympus). The MetaMorph software (Molecular Devices) was used for quantification.
We performed behavioral studies to assess motor function as previously described (22, 33). For the tail suspension test, each mouse was hung from its tail at a height of 80 cm for 120 s, and the clasping duration was scored. For the hanging wire test, each mouse was placed on the wire lid of a conventional housing cage, and the lid was turned upside down. The latency from the beginning of the test until the mouse stood with at least two limbs on the lid was recorded. The animals made three attempts to stand for a maximum of 180 s per trial, and the longest latency was recorded. The rotarod apparatus (accelerating model 47600; Ugo Basile) was set to an initial speed of 4 rpm, and the acceleration was increased by 32 rpm every 25-30 s. Scores were registered every two days, and three independent tests were performed at each measurement. Uniform conditions were carefully maintained for each test, with a rest time of 1 h between trials. Each test was limited to 300 s.
Sample sizes were determined using the G*Power software (with α = 0.05 and a power of 0.8). In general, statistical methods were not used to recalculate or pre-determine sample sizes. The variance was similar within comparable experimental groups. Individuals performing the experiments were blinded to the identity of the experimental groups until the end of the data collection and analysis for at least one independent experiment. In cases where more than two groups were compared, one-way analysis of variance (ANOVA) was used, followed by Tukey’s HSD test. All statistical analyses were performed using the GraphPad Prism software (version 7.0). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered to be significant.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1A2C3006875, 2020R1A2C3006734). This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare and MSIT, Republic of Korea (HU20C0345).
The authors have no conflicting interests.
Human plasma information for control subjects and subjects with amyotrophic lateral sclerosis (ALS)