Microarray analysis of the cellular pathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS

Abstract

The cellular pathways of motor neuronal injury have been investigated in the SOD1 G93A murine model of familial amyotrophic lateral sclerosis (ALS) using laser-capture microdissection and microarray analysis. The advantages of this study include the following: analysis of changes specifically in motor neurons (MNs), while still detecting effects of interactions with neighboring cells; the ability to profile changes during disease progression, an approach not possible in human ALS; and the use of transgenic mice bred on a homogeneous genetic background, eliminating the confounding effects arising from a mixed genetic background. By using this rigorous approach, novel changes in key cellular pathways have been detected at both the presymptomatic and late stages, which have been validated by quantitative reverse transcription-PCR. At the presymptomatic stage (60 d), MNs extracted from SOD1 G93A mice show a significant increase in expression of genes subserving both transcriptional and translational functions, as well as lipid and carbohydrate metabolism, mitochondrial preprotein translocation, and respiratory chain function, suggesting activation of a strong cellular adaptive response. Mice 90 d old still show upregulation of genes involved in carbohydrate metabolism, whereas transcription and mRNA processing genes begin to show downregulation. Late in the disease course (120 d), important findings include the following: marked transcriptional repression, with downregulation of multiple transcripts involved in transcriptional and metabolic functions; upregulation of complement system components; and increased expression of key cyclins involved in cell-cycle regulation. The changes described in the motor neuron transcriptome evolving during the disease course highlight potential novel targets for neuroprotective therapeutic intervention.

Figure 1.

Q-PCR results. A, Significant upregulation of necdin at 60 d (p = 0.04), which is downregulated at the late stage of the disease (p = 0.003). B, Consistent upregulation of cyclin I throughout the pathology (60 d, p = 0.05; 90 d, p = 0.1; 120 d, p = 0.03). C, Consistent downregulation of plexin domain containing 1 (60 d, p = 0.02; 90 d, p = 0.003; 120 d, p = 0.001). Error bars indicate SD. Tg, Transgenic; N-Tg, nontransgenic. *p ≤ 0.05; **p ≤ 0.01.

Figure 2.

Gene ontology classification showing the number of transcripts upregulated and downregulated per category throughout the pathology. Bars on the left indicate the number of upregulated genes, and bars on the right indicate the number of downregulated genes, for each category. The transcripts most affected by the progression of the disease are those encoding for mitochondrial proteins (mito. proteins), proteins presenting oxidoreductase activity, and proteins related to the transcriptional function. These classes undergo a complete inversion in their regulation with the progression of the disease.

Figure 3.

A, Motor neurons isolated from G93A mice at 60 d show upregulation in several classes of genes delineating what is likely to be happening in these cells under stress. There is upregulation of the transcriptional machinery, along with upregulation of translation-related ribosomal and folding proteins, as the motor neuron attempts to compensate for the ongoing cellular stress. All these mechanisms require ATP, provoking a massive increase in the work load of mitochondria, leading to upregulation of carbohydrate metabolism and respiratory chain activity, which in turn causes increased ROS production. The observed imbalance among the subunits forming the ATP synthase complex, shown by downregulation of the δ subunit, will generate additional oxidative stress, with consequent production of oxidized proteins. These are then ubiquitinated and targeted for proteasomal degradation. B, Over time, the accumulation of damaged proteins and ROS is likely to cause a general collapse in cellular functioning, leading to downregulation of the compensatory pathways previously activated and leaving the cell with decreased energy and protein turnover. The cell increases protein degradation functions, with activation of the lysosomal machinery. Production and secretion of some subunits of the complement cascade are important signals of cellular stress for neighboring cells. The final abortive attempt at survival comes through activation of the cell cycle, with upregulation of cyclin L1 (involved in the transition from the quiescent state, G₀, to the first phase of the cell cycle, G₁) and cyclins D2 and E2 (involved in the progression of the cell cycle through G₁ phase). The upregulation of cyclin I suggests that motor neurons are trying to exercise negative control on the transition between the G₁ and S phase to prevent the abnormal progression through the cell cycle. Atf4, Activating transcription factor 4; Atp5a1, ATP synthase F1 complex α1 subunit; Atp5d, ATP synthase F1 complex δ subunit; Cct4, chaperonin subunit 4; Ctsz, cathepsin-Z; Eef1, eukaryotic translation elongation factor 1; Eif3, eukaryotic translation initiation factor 3; Hsp, heat shock protein; Lyz, lysozyme; Lzp-s, P-lysozyme structural; Mdh1, malate dehydrogenase 1; Ndn, necdin; Psmc6, proteasome 26S; Rpl, ribosomal protein L; Sdha, succinate dehydrogenase complex subunit A; Taf9, transcription activator factor 9; Tcerg1, transcription elongation regulator 1 (CA150); Tgfb1i4, transforming growth factor β1-induced transcript 4; Ube1c, ubiquitin-activating enzyme E1C; Uble1b, ubiquitin-like activating enzyme E1B; Usp36, ubiquitin-specific preotease 36; H⁺, hydrogen ion; TCA, tricarboxylic acid.