LncRNA levels in the central nervous system as novel potential players and biomarkers in amyotrophic lateral sclerosis

Abstract

Research in amyotrophic lateral sclerosis (ALS) faces major burdens, including the urgent need for sensitive and specific biomarkers, the identification of novel and effective therapeutic targets and a deeper understanding of the mechanisms driving the disease. In this line, long non-coding RNAs (lncRNAs) have emerged as promising candidates due to their regulatory role in a variety of important biological processes such as RNA metabolism, neuroinflammation, apoptosis or proteostasis. This study aims to elucidate the expression profile of 14 lncRNAs in both the SOD1^G93A mouse model and ALS patients. Different stages of the disease (presymptomatic, symptomatic and terminal) and 3 regions of the central nervous system (CNS) differentially affected by ALS (spinal cord, brainstem and frontal cortex) were included in the experimental design. In SOD1^G93A mice, all 14 lncRNAs exhibited differential expression patterns influenced by sex, age, and region, except for Malat1, Neat1, and H19, which displayed consistent expression patterns (Malat1 was decreased, while Neat1 and H19 were increased). These patterns were most prominent in the spinal cord, where lncRNAs were overall down-regulated. In contrast, in the brainstem and frontal cortex, lncRNAs were predominantly up-regulated. Notably, Gas5 expression levels in frontal cortex and spinal cord at the terminal stage correlated with the onset and progression of motor coordination and strength decline. Additionally, three lncRNAs (Gas5, Neat1 and Myoparr) were found to significantly correlate with survival. In human ALS samples, increased levels of NEAT1 and SNHG16 were observed in the brainstem, and of MEG3 and H19 in the frontal cortex, whereas MALAT1 levels were decreased in frontal cortex. In conclusion, this work supports lncRNAs as promising candidates as novel players and potential biomarkers in ALS and highlights SOD1^G93A mice as a good model to study lncRNAs in the CNS in the context of this disease.

Keywords: ALS; Gas5; H19; Malat1; Myoparr; Neat1.

Fig. 1

Selection of lncRNAs potentially involved in ALS. (A) Selection criteria (B) LncRNAs selected from the study and associated molecular mechanisms.

Fig. 2

Differentially expressed lncRNAs in the central nervous system of SOD1^G93A ALS mice. (A) Volcano plot of differentially expressed genes identified between SOD1^G93A mice and age-matched control group. Each point represents the fold-change of ALS mouse model at a given stage (pre-symptomatic, symptomatic or late symptomatic) and tissue (spinal cord, brainstem and frontal cortex). The blue dots denote down-regulated gene expression, the red dots denote up-regulated gene expression. (B–E) Venn diagrams of down- and up-regulated lncRNAs taking into account sex-mixed groups of ALS and WT mice (B down-regulated, C up-regulated) and separating by sex (D down-regulated, E up-regulated). Yellow, green and purple colours correspond to the spinal cord, brainstem and frontal cortex, respectively. N = 24 (spinal cord) and N = 20 (brainstem and frontal cortex) per stage, balanced in sex and genotype. Results shown were obtained by RT-qPCR; supplemental information for this figure can be found in Supplementary Fig. 1 and Supplementary Table 7.

Fig. 3

LncRNA expression pattern in the central nervous system of SOD1^G93A ALS mice. Heat map of lncRNA fold change of male (A) and female (B) SOD1^G93A mice in different regions of the central nervous system. LncRNA fold change is expressed as a value rated from 0 to >3, calculated as the ratio of RNA expression levels by RT-qPCR in ALS mice compared to their age-matched WT littermates. Xist expression is absent in males, as its transcription is restricted to individuals with two X chromosomes. N = 24 (spinal cord) and N = 20 (brainstem and frontal cortex) per stage, balanced in sex and genotype. Supplemental information for this figure can be found in Supplementary Table 7. PS: presymptomatic stage (P60), ES: symptomatic stage (P90-P100), LS: late symptomatic stage (P120). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 4

LncRNAs Gas5 and Myoparr as potential modifiers of ALS progression in SOD1^G93A mice. (A,B)Gas5 levels in the spinal cord of females at the humane endpoint correlate with muscle strength failure onset (A) and disease duration (B). (C,D)Gas5 levels in the frontal cortex of terminal SOD1^G93A males correlates with motor coordination and balance failure onset (C) and disease duration (D). (E)Gas5 levels in the brainstem at the humane endpoint correlate with SOD1^G93A mice survival. (F)Myoparr levels in the frontal cortex of terminal SOD1^G93A males correlates with survival. For this experiment, N = 23 SOD1^G93A mice (n = 11 males, n = 12 females) at the humane endpoint. Results were obtained by RT-qPCR.

Fig. 5

Differential lncRNA expression in the brainstem and frontal cortex of sALS patients. (A,C) Statistically significant lncRNA fold changes in the brainstem (A) and frontal cortex (C) of sALS patients vs. controls. (B) ROC curve for NEAT1 and SNHG16 ΔCt in brainstem. (D) ROC curve for MEG3, MALAT1 and H19 ΔCt in frontal cortex. (E) Descriptive statistics for ROC curves presented in (B) and (D). Data were obtained by RT-qPCR and are represented as mean ± SD. ROC curve plots discriminate between sALS patients and controls. ∗p < 0.05, ∗∗p < 0.01.