**6.2 Gene expression profiling of laser capture microdissection isolated cell types**

In order to overcome the limitations of using mixed cell population samples, dissection of single cells from complex tissues using LCM has been applied to identify the contribution of different cell types to the degenerative process occurring in ALS.

Insights Arising from Gene Expression Profiling in Amyotrophic Lateral Sclerosis 49

expression B cell translocation gene-1 (*Btg1*), growth differentiation factor-5 (*Gdf5*) and

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

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.

myogenic factor-6 (*Myf6*) are potent activators of new fibre formation.

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

research in ALS (Ferraiuolo et al 2011b).

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

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

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 were identified (Figure 2).

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 the murine model.

#### **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 expression B cell translocation gene-1 (*Btg1*), growth differentiation factor-5 (*Gdf5*) and myogenic factor-6 (*Myf6*) are potent activators of new fibre formation.
