Genetic biomarkers for ALS disease in transgenic SOD1(G93A) mice

Abstract

The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1(G93A) mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1(G93A) mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1(G93A) mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies.

Figure 1. Transcriptional expression levels of the sixteen genes varied significantly throughout disease progression in transgenic SOD1^G93A mice.

Representative graphs showing the fold change in transcriptional levels of the seventeen genes tested in the skeletal muscle of transgenic SOD1^G93A mice throughout disease progression with respect to the early symptomatic stage. Age-matched wild type mice were used as controls in each stage of the disease: early symptomatic (60 days, grey bar), symptomatic (90 days, blue bar) and terminal (120 days, pale blue bar) stages. The highest transcriptional expression levels were found in Ankrd1 and Col19a1, which at the terminal stage were almost 27 and 18 times higher than those observed at the early symptomatic stage, respectively. A significant upregulation of transcriptional levels was found in all of the genes, except for Calm1, despite its irregular profile pattern throughout disease progression.

Figure 2. Transcriptional SOD1^G93A fold change in muscle biopsy samples.

The transcriptional expression levels of SOD1^G93A were measured in all the samples obtained from the muscle biopsies corresponding to early symptomatic (first biopsy), symptomatic (second biopsy) and terminal stages (third biopsy). No statistical differences were found in SOD1^G93A levels along disease progression. SOD1^G93A fold change in the symptomatic and terminal stages was calculated respect to the relative expression found in all the muscle samples extracted at the early symptomatic stage.

Figure 3. Eleven genes were related to the denervation process.

Fold change variation in the transcriptional levels of seventeen genes in the skeletal muscle of 74-day-old denervated mice. Wild type mice aged for 60 days were used as controls. Among the seventeen genes tested, the transcriptional levels of Ankrd1, Rrad, Myog, Mt2, Myod1, Sln, Myf5, Pax7, Nogo A, Impa1 and Fbox32 varied significantly in denervated mice compared to control mice. Fbox32 was the only gene that displayed a downregulated transcription level in denervated mice, which is probably due to its role in compensating for denervation-induced muscle atrophy via an internal mechanism.

Figure 4. Mef2c, Gsr, Col19a1, Calm1 and Snx10 are potential genetic biomarkers of longevity in ALS.

Linear correlation graphs of five potential genetic biomarkers of longevity. The graph shows the linear relation between the fold change in transcriptional levels of these genes throughout disease progression in skeletal muscle biopsies at the early symptomatic stage and the longevity of the animals at the terminal stage. The transcriptional levels of these genes can predict longevity in transgenic SOD1^G93A mice.