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

As with the mouse models, in order to determine those genes differentially expressed in the vulnerable cell type, LCM has been used in post-mortem material to isolate the motor neurones from the spinal cord.

Motor neurones isolated from SALS cases and neurologically normal controls were shown to have distinct gene expression profiles compared to those generated from ventral horn homogenates, particularly with respect to those genes down-regulated in motor neurones (Jiang et al 2005). The motor neurones showed differential expression of genes associated with the cytoskeleton and evidence of decreased transcription, whilst cell death-associated genes and those involved in cell signalling were increased (Figure 3). In addition, cell cycle related genes were also reported as dysregulated, supporting the theory that inappropriate activation of the cell cycle in these post-mitotic cells leads to cell death. A follow-up study demonstrated that expression of several of these genes also correlated with pathological markers, such as phosphorylated neurofilament and ubiquitinated protein accumulations as well as motor neurone loss (Jiang et al 2007).

Gene expression profiling of isolated motor neurones has also been performed on ALS cases which carry genetic mutations in the *SOD1* and chromatin modifying protein 2B (*CHMP2B*) genes (Cox et al 2010; Kirby et al 2011). Motor neurones from *SOD1*-related cases of ALS showed increases in genes in the protein kinase B/phosphatidylinositol-3 kinase (AKT/PI3K) cell survival pathway, with concomitant decreases in negative regulators such as phophastase and tensin homologue (*PTEN*) (Kirby et al 2011) (Figure 3). Further work demonstrated that inhibition of PTEN led to increased activation of the AKT/PI3K pathway and increased neuronal survival in cell models including primary motor neurone cultures. Thus, activation of the AKT/PI3K pathway was proposed as a candidate for future therapeutic strategies.

The transcriptional profiles from motor neurones isolated from the *CHMP2B*-related ALS cases were distinct from those in *SOD1*-related cases (Cox et al 2010). These motor neurones showed dysregulation of genes involved in the classical and p38 MAPK signalling pathways, gene changes predicted to reflect reduced autophagy and repression of translation (Figure 3). The functional implication of *CHMP2B* mutations on cellular mechanisms was demonstrated by the presence of large cytoplasmic vacuoles and impairment of autophagy in a cellular model transfected with mutant *CHMP2B*, consistent with the microarray findings (Cox et al 2010). Interestingly, differential expression of genes encoding proteins responsible for calcium handling and cell cycle genes, as well as those genes involved in transcription, signalling and metabolism, was detected in both genetic subtypes.
