Nusinersen corrects L-arginine deficiency in the cerebrospinal fluid of patients with severe spinal muscular atrophy

Abstract

Spinal Muscular Atrophy (SMA) is a progressive neuromuscular disorder caused by homozygous loss of the survival motor neuron 1 (SMN1) gene, leading to reduced SMN protein expression. Increasing evidence implicates neurotransmission deficits in the pathophysiology of SMA. In particular, alterations in neuroactive amino acids involved in glutamatergic neurotransmission have recently been identified in both the cerebrospinal fluid (CSF) of SMApatients and the spinal cord of SMNΔ7 mouse models. L-arginine, a precursor of nitric oxide, plays a critical role in glutamatergic receptor signalling, influencing neurotransmitter release, synaptic plasticity, and neuroprotection. However, it remains unclear whether SMN deficiency affects L-arginine metabolism in SMA. To address this, we used high-performance liquid chromatography to investigate whether SMN deficiency alters L-arginine homeostasis in the central nervous system of SMNΔ7 mice and in the CSF of SMA patients with varying disease severity, both before and after treatment with the SMN-inducing therapy Nusinersen. Notably, we observed significantly reduced L-arginine levels in the brainstem and spinal cord of symptomatic SMA mice compared to age-matched wild-type littermates. Consistent with these findings, we revealed lower L-arginine levels in severe SMA1 patients compared to milder SMA2 and SMA3 patients and healthy controls, enhancing the translational strength of our findings. Importantly, Nusinersen-mediated SMN upregulation fully restored L-arginine homeostasis in the CSF of severe SMA1 patients. In conclusion, our results demonstrate a dysregulation of L-arginine in SMA and highlight a role for SMN-enhancing therapies in restoring neurochemical alterations observed in patients with this neurodegenerative disease.

Keywords: Cereberal spinal fluid; L-arginine; Nusinersen; SMNΔ7 mice; Spinal muscular atrophy.

Figure 1.. Decreased L-arginine levels in the brainstem and spinal cord of SMA mice at overt-symptomatic stage of the disease.

(a) Representative chromatograms obtained from spinal cord of wild-type (W) mice at post-natal day (P) 12 with L-arginine peak magnification. (b) Schematic representation of time points (P1, P5, and P12) corresponding to key phases in early postnatal development and disease progression in SMNΔ7 mice at which mice have been sacrificed in the present study (created with Biorender.com);. Levels of L-arginine in brainstem (c), spinal cord (d), cortex (e) and cerebellum (f) in W and SMA (S) mice at P1, P5, and P12. Data are expressed as nmol/mg of protein and are shown as violin plots representing the median with interquartile range. *p < 0.05 (Mann-Whitney test). Dots represent values from each mouse analyzed, n=7/group.

Figure 2.. The concentration of L-arginine in cerebrospinal fluid is influenced by the disease severity of spinal muscular atrophy patients.

(a) Representative chromatogram obtained from the cerebrospinal fluid (CSF) of a 1-year-old patient showing the peak of L-arginine; (b) Levels of L-arginine (μM) in the indicated cohorts of drug-free SMA1 (n=29), SMA2 (n=19) and SMA3 (n=13) patients as well as control individuals (n=7). Data are shown as violin plots representing the median with interquartile range (IQR). For statistical analysis data were natural log transformed and ANCOVA considering age and sex as confounding factors was performed, *p<0.05, **p<0.01.

Figure 3.. L-arginine displays an age-dependent reduction in the CSF of SMA2 patients.

Correlation between cerebrospinal fluid (CSF) concentrations of L-arginine (μM) and age (years) in SMA1 (n=29), SMA2 (n=18), SMA3 (n=10) patients, and age-matched controls (CTRL) (n=7). Each dot represents an individual subject. Lines and shadows represent the best fit line and its 95% confidence interval, respectively. Non-parametric Spearman correlation: *p < 0.05.

Figure 4:. Nusinersen selectively increases L-arginine concentrations in the cerebrospinal fluid of patients with SMA1.

(a) Schematic representation of the timeline of intrathecal Nusinersen administration and cerebrospinal fluid (CSF) collection in SMA patients including loading and maintenance dose phases with sample collection points at T0 and T302 (created with Biorender.com); (b) Spaghetti plots represent L-arginine concentrations (μM) in CSF of SMA1 (n=14), SMA2 (n=14), SMA3 (n=10) patients at baseline (T0) and after 302 days of Nusinersen treatment (T302). L-arginine concentrations (μM) in CSF of controls (Ctrl) is also reported.Each dot indicates an individual subject. Paired Wilcoxon test, *p < 0.05.