**2. What is gene expression profiling?**

The primary application of gene expression profiling is to use a glass slide containing oligonucleotides or cDNA sequences, termed a microarray, to quantify the amount of individual RNA transcripts that are present in a particular cell or tissue. RNA is extracted, fluorescently labelled and then hybridized to the microarray. A linear amplification step can also be performed prior to labelling to ensure sufficient RNA is generated from low yields of RNA extracted from small sample sizes. The quantity of labelled RNA binding to each oligonucleotide or cDNA sequence on the microarray determines the intensity of fluorescence at that location and thus allows quantification of the RNA transcripts in the initial sample (Stoughton 2005).

Comparison of the expression of transcripts between sample groups allows identification of transcripts which are differentially expressed between the groups. The large number of genes simultaneously interrogated by a microarray means that the resulting data are amenable to study the expression of inter-related clusters of genes such as those involved in known functional categories or specific pathways. Changes in expression level of a particular gene do not take into account regulation at the level of translation. However, the functional impact of a differentially expressed gene can be subsequently established by assaying the encoded protein expression or function.

Insights Arising from Gene Expression Profiling in Amyotrophic Lateral Sclerosis 43

Gene expression profiling of cell lines transfected with ALS associated genes provides a genetically homogenous cell population uncontaminated by non-neuronal astrocytes and other types of glial cells, which are present in the central nervous system (CNS). Furthermore, environmental conditions can easily be manipulated and tightly controlled *in vitro* so as to reduce the impact of external confounding factors on gene expression. The limitations of this type of model system include the fact that NSC-34 cell lines (or other neuronal cell lines) are continually dividing cells, rather than post-mitotic cells and they are unable to mirror the effects of cellular interactions that occur between the different cell

Primary neuronal and astrocytic cells can be isolated from embryonic mice and short-term cultures generated for microarray analysis. These cells more closely mirror those present in the CNS, though the primary neuronal cultures, as they are post-mitotic cells, have a limited lifespan of 7-10 days. In contrast, cultured primary astrocytes are able to proliferate in culture. Whilst co-cultures or separated co-cultures allow a degree of interaction between the two cell types, these types of mixed cultures have not yet been used for microarray analysis in ALS.

Transgenic mice expressing mutant forms of ALS-related genes provide a source of RNA for microarray analysis. For investigating the mechanisms of *SOD1*-related neurodegeneration, mice over-expressing the human p.G93A or mouse p.G86R mutant forms of the SOD1 protein (SOD1G93A or SOD1G86R) have been used as they develop an age dependent neuromuscular condition; the motor function symptoms and histopathological features have been extensively characterised and resemble those observed in both *SOD1*-related ALS and classical ALS patients (Gurney et al 1994; Ripps et al 1995). In contrast, over-expression of wild-type human SOD1 (SOD1WT) does not produce an overt motor phenotype, supporting a toxic gain of function by the mutant SOD1 protein as the mechanism by which the mutant proteins cause cell death. In contrast, mouse models over-expressing either wild-type or mutant TDP-43 show

One of the major advantages of using animal models for microarray analysis is the ability to examine animals at different ages in order to investigate the progression of disease, an approach that is unattainable in human post-mortem tissue. Valuable insights regarding onset of disease can be established in pre-symptomatic and early symptomatic disease stages since these represent time points at which the identification of key novel targets for therapeutic intervention could be best placed to rescue vulnerable neuronal cell populations before the development of irreversible neuronal injury (Ferraiuolo et al 2007). In addition, sampling of specific cell types from the CNS allow gene expression changes to be identified

Backcrossing of the SOD1WT and SOD1G93A mice with C57Bl6 mice has led to the formation of SOD1WT and SOD1G93A mice on a homogeneous background (Ferraiuolo et al 2007). The use of these mice for microarray analysis and the use of non-transgenic littermates as controls have proven effective in reducing inter-individual genetic variability to ensure the

Human post-mortem brain and spinal cord specimens derived from clinically and pathologically confirmed cases of ALS can be used in comparisons with age, gender and

a neurodegenerative phenotype (Igaz et al 2011; Stallings et al 2010).

which include the effects of interactions with neighbouring cells.

generation of consistent and reliable gene expression data.

**4.3 Human post-mortem material** 

populations *in situ* (Kirby et al 2011).

**4.2 Animal models of ALS** 

The advent of next generation sequencing, and specifically the sequencing of all RNA molecules in a quantitative manner, has recently become an alternative, though expensive method for measuring levels of gene expression. However, this has the potential to add further knowledge and value to the application of gene expression profiling to disease.
