**7. Results from use of human post-mortem material**

48 Amyotrophic Lateral Sclerosis

Several studies have determined the changes in gene expression occurring in motor neurones isolated from the spinal cord of mutant SOD1G93A mice at different stages during the disease; from the pre-symptomatic stage to paralysis (Ferraiuolo et al 2007; Perrin et al 2005). The first report described transcriptional analysis of motor neurones isolated from SOD1G93A mice bred on a mixed background and no differentially expressed genes were detected in the pre-symptomatic mice (Perrin et al 2005). However, in contrast to the whole tissue homogenates, motor neurones did not show activation of apoptotic genes, suggesting

In the second publication, microarray analysis was carried out on SOD1G93A mice bred on a homogeneous background; this enabled important changes in the motor neurones at the pre-symptomatic stage of disease, mainly involved in carbohydrate metabolism and transcription, to be detected (Ferraiuolo et al 2007). The upregulation of transcripts encoding proteins involved in the energy production pathway, i.e. tricarboxylic acid cycle and respiratory chain, suggested that motor neurones were trying to compensate for their increased energy needs in response to ongoing stress. At the late stage of disease, increased expression of transcripts involved in reactivation of the cell cycle (as an alternative pathway of cell death), and complement activation (a mechanism through which motor neurones can attract cells from the immune system), and down-regulation of transcription-related genes

To complement the gene expression profiling of motor neurones, astrocytes isolated from SOD1G93A mice at the pre-symptomatic stage of disease were isolated and used for microarray analysis (Ferraiuolo et al 2011a). This enabled the cross-talk between the motor neurones and astrocytes at this very early time point to be interrogated. Interestingly, astrocytes displayed a marked impairment of carbohydrate metabolism (Figure 2). Comparing the expression profiles of the two cell types from the same SOD1G93A mice highlighted that the metabolic impairment observed in motor neurones could derive from the lack of provision of substrates, i.e. lactate, from the astrocytes, and led to the conclusion that the lactate shuttle (the mechanism through which motor neurones and astrocytes combine metabolism and signalling through lactate and glutamate), is impaired. In addition, the activation of an important neuronal cell death pathway involving p75 and its ligand pro nerve growth factor (proNGF) was established. Gene expression profiling of SOD1G93A astrocytes demonstrated that these cells expressed high levels of *Ngf,* while the motor neurones over-expressed the p75 receptor. *In vitro* data confirmed the dysregulation of both pathways and preliminary data from human ALS biosamples supported these findings from

**6.3 Gene expression profiling of peripheral tissue from mouse models** 

Microarray technology has also been applied to peripheral tissues from the SOD1G86R mouse model (Gonzalez de Aguilar et al 2008). Profiling of the skeletal muscles revealed that the major expression changes happen at onset of disease, when muscles are activating pathways involved in detoxification and regeneration, but also cell death and tissue degradation. These findings revealed that while motor neurones are degenerating, muscles are undergoing major remodelling trying to compensate for muscle damage with new myogenesis. Whilst over-expression of transcripts such as cyclin-dependent kinase inhibitor-1A (*Cdkn1a*) and growth arrest-and DNA damage-inducible gene-45 (*Gadd45*) could be mediating apoptosis of myofibres resulting in muscle atrophy, increased

that cell death signals derive from other cell types in the spinal cord (Figure 2).

were identified (Figure 2).

the murine model.

### **7.1 Gene expression profiling of mixed cell type CNS samples**

Multiple studies have utilised gene expression profiling of post mortem mixed cell samples from ALS patients and controls; these have either focused on using samples from the motor cortex (Lederer et al 2007; Wang et al 2006) or from the spinal cord (Dangond et al 2004; Malaspina et al 2001; Offen et al 2009). The majority used sporadic ALS (SALS) cases, though Dangond et al also sampled two FALS cases, one of which carried an *SOD1* mutation (Dangond et al 2004). Despite the different tissues profiled and the different platforms utilised, the studies showed some consistent results: All of the studies recorded altered gene expression related to inflammation and Malaspina and colleagues detected an increase in glial fibrillary acidic protein (*GFAP*), indicating active astrogliosis (Figure 3). In addition, a number of the studies discovered differential expression related to cytoskeleton function, protein processing and the antioxidant response, in agreement with other lines of research in ALS (Ferraiuolo et al 2011b).

Fig. 3. Summary of prominent pathways arising from GEP of Human Tissue. Important changes in the transcriptome have been highlighted by green labels; yellow stars indicate up-regulation, red stars indicate down-regulation. Blue squares outline functional consequences of changes in the transcriptome. Further details can be found in the text.

Insights Arising from Gene Expression Profiling in Amyotrophic Lateral Sclerosis 51

differential gene expression. This work also provided evidence that peripheral blood is a valuable medium for studying ALS. In addition, there was a correlation with the CNS tissue studies, as the blood in ALS patients showed a decrease in genes associated with protein processing and RNA post-transcriptional modification, as well as increases in genes

A more recent study performed gene expression profiling on peripheral blood mononuclear cells from patients with SALS (Zhang et al 2011). These cells showed an up-regulation of genes associated with immune activation in response to lipopolysaccharide (LPS), which correlated with an elevation of plasma LPS. Unfortunately, no correlation between

Transcriptional profiles of fibroblasts from patients with SALS, FALS and controls have been shown to be informative in distinguishing the different genetic variants from each other, as well as from SALS patients, by their gene expression profiles and specifically the level of alternative splicing (Highley et al 2011). The FALS samples were derived from patients with mutations in *SOD1* and *TARDBP*. Using microarrays which interrogate every exon of every gene, it was demonstrated that there was a significant amount of aberrant splicing in the samples with a *TARDBP* mutation which was replicated to a lesser degree in the SALS samples and virtually absent in the samples with a *SOD1* mutation (Figure 3). In support of this, other work has identified aberrant splicing in cell and animal models with

The advent of next generation sequencing has evolved to enable quantitative parallel sequencing of RNA transcripts from isolated cells and tissues. There are a number of advantages of next generation RNA sequencing over the microarray platform which in general extend from the fact that there is no reliance on the pre-designed probes which are present on the microarrays. In contrast, the methodology aims to sequence every base of every transcript of RNA. This leads to a better detection rate of known transcripts and splicing events and the detection of RNA transcripts (both coding and non-coding) which have not previously been described and therefore have no specific probe (Sultan et al 2008). As there is no reliance on probes, the problem of cross hybridisation is avoided. In addition, because each base in each transcript is sequenced, as well as providing information about expression level and alternative splicing, the sequencing also provides information about sequence variability within the RNA (Wang et al 2009). The biggest challenge to RNA sequencing, however, is the analysis of the large amounts of data produced which is substantially more than the read out of even the most comprehensive microarray. Not least among these challenges is the problem of mapping the RNA sequences to the genome, as in contrast to DNA sequencing, the absence of introns can lead to substantial difficulties

This chapter has focused on the mRNA that is translated into protein. However, although 90% of eukaryotic genomic DNA is transcribed, only 1-2% actually encodes protein. The vast majority of transcribed material is comprised of non-coding RNA (ncRNA) and there is

expression of these genes and disease progression was provided.

depletion of TDP-43 (Polymenidou et al 2011; Tollervey et al 2011).

**9. The future of gene expression profiling in ALS** 

associated with inflammation.

**9.1 RNA sequencing (RNA-seq)** 

(Sutherland et al 2011).

**9.2 Role of microRNAs in ALS** 
