Gene expression changes in spinal motoneurons of the SOD1(G93A) transgenic model for ALS after treatment with G-CSF

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3-5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1(G93A) mice, the most frequently studied animal model for ALS, with and without G-CSF treatment.

Results: Motoneurons from SOD1(G93A) mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1(G93A) motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12.

Conclusions: Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1(G93A) motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.

Keywords: ALS; G-CSF; gene expression; laser microdissection; motoneuron; mouse model; neurodegeneration.

Figure 1

Study design. (A) Time course of the study. Given is the disease course in SOD1G93A mice, time of treatment and time of sacrifice for all experimental groups. (B) Flow chart for analytical steps for analysis.

Figure 2

Canonical plot shows dispersion of treated animals off the non-treated groups. Multivariate analyses have been conducted with genes having a standard deviation greater than 1.2 (734 transcripts). (A) Canonical plot shows that symptomatic SOD-1 mice (red dots) are distant from other groups, including asymptomatic SOD1 mice (blue dots) and when considered as unknown, SOD-1 mice treated with G-CSF (green dots) show a strong dispersion and tend to be close to wild types at 11 weeks (gray dots) or 15 weeks of age (black dots). (B) After G-CSF treatment, discriminant analysis classified only three samples of SOD1 motorneurons as symptomatic motor neurons. One sample was considered has presymptomatic and three as wild type motor neurons.

Figure 3

G-CSF treatment modulates transgene-related gene expression changes. (A) Chart showing the number of differentially regulated genes after G-CSF treatment in the motoneurons of SOD1 mice. (B–C) Given is the relative expression of the 30 most significantly modified transcripts re-adjusted by G-CSF treatment and previously found downregulated (B) or upregulated (C) in symptomatic SOD1 mice.

Figure 4

Validation of the expression of selected transcripts by qPCR. (A–D) Expression levels from microarray analyses of Bmpr1a (A), CNOT10 (B), CTSB (C) and PRSS12 (D) at the symptomatic disease stage. (E–H) Expression levels derived from quantitative PCR of Bmpr1a (E), CNOT10 (F), CTSB (G) and PRSS12 (H) at the symptomatic disease stage. Data are presented as mean ± standard variation of the mean. ^*p < 0.05 (ANOVA, followed by multicomparison t-test).