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

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

50 Amyotrophic Lateral Sclerosis

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

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

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

Gene expression profiling has been conducted on blood and fibroblasts from ALS patients (Highley et al 2011; Saris et al 2009; Zhang et al 2011). Microarray analysis of SALS and control blood samples was followed by hierarchical clustering of all genes found to be significantly expressed in all samples after normalisation (Saris et al 2009). The method identified five clusters; two of which were able to differentiate between ALS and control samples. These clusters were replicated in a further two cohorts of patients and controls, demonstrating that such an analysis, which takes into account the interdependence of gene expression, is a means of reducing the false negative rate when subsequently detecting

**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.

therapeutic strategies.

subtypes.

well as motor neurone loss (Jiang et al 2007).

**8. Results from use of human peripheral tissue** 

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 (Sutherland et al 2011).

### **9.2 Role of microRNAs in ALS**

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

Insights Arising from Gene Expression Profiling in Amyotrophic Lateral Sclerosis 53

has begun to allow the distinctions between genetic and sporadic disease to be differentiated as well as providing further candidates for therapeutic approaches. In the future, gene expression profiling of larger, consistently collected patient samples has the potential to generate robust and reliable prognostic and diagnostic biomarkers, which will ultimately be

PJS and JK are funded by the European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement 259867 and by the Motor Neurone

Aguirre T, Van Den Bosch L, Goetschalckx K, Tilkin P, Mathijs G, et al. 1998. Increased

Booij BB, Lindahl T, Wetterberg P, Skaane NV, Saebo S, et al. 2011. A gene expression

Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, et al. 2005. Genome-wide expression

Boutahar N, Wierinckx A, Camdessanche JP, Antoine JC, Reynaud E, et al. 2011. Differential

Budini M, Buratti E. 2011. TDP-43 Autoregulation: Implications for Disease. *J Mol Neurosci* Buratti E, De Conti L, Stuani C, Romano M, Baralle M, Baralle F. 2010. Nuclear factor TDP-

Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, et al. 1992. Neuroblastoma x

Cox LE, Ferraiuolo L, Goodall EF, Heath PR, Higginbottom A, et al. 2010. Mutations in

Dadon-Nachum M, Melamed E, Offen D. 2011. The "dying-back" phenomenon of motor

Dangond F, Hwang D, Camelo S, Pasinelli P, Frosch MP, et al. 2004. Molecular signature of

Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, et al. 2008. Induced

Duan W, Li X, Shi J, Guo Y, Li Z, Li C. 2010. Mutant TAR DNA-binding protein-43 induces

oxidative injury in motor neuron-like cell. *Neuroscience* 169:1621-9

43 can affect selected microRNA levels. *FEBS J* 277:2268-81

sensitivity of fibroblasts from amyotrophic lateral sclerosis patients to oxidative

pattern in blood for the early detection of Alzheimer's disease. *J Alzheimers Dis*

profiling of human blood reveals biomarkers for Huntington's disease. *Proc Natl* 

effect of oxidative or excitotoxic stress on the transcriptional profile of amyotrophic lateral sclerosis-linked mutant SOD1 cultured neurons. *J Neurosci Res* 89:1439-50

spinal cord (NSC) hybrid cell lines resemble developing motor neurons. *Dev Dyn*

CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS).

late-stage human ALS revealed by expression profiling of postmortem spinal cord

pluripotent stem cells generated from patients with ALS can be differentiated into

Disease Association (MNDA). EFG is also funded by the MNDA.

applicable for use in the clinic.

stress. *Ann Neurol* 43:452-7

*Acad Sci U S A* 102:11023-8

**11. Acknowledgments** 

23:109-19

194:209-21

*PLoS ONE* 5:e9872

neurons in ALS. *J Mol Neurosci* 43:470-7

gray matter. *Physiol Genomics* 16:229-39

motor neurons. *Science* 321:1218-21

**12. References** 

increasing evidence to support functional roles for at least a subset of these transcripts (Kaikkonen et al 2011). There are broadly two types of ncRNA, infrastructural (including transfer RNA and small nuclear RNA) and regulatory RNA (including microRNA, Piwiinteracting RNA and small interfering RNA). The function of ncRNAs remains largely unknown. However, research into microRNA (miRNA) has led the field in recent years. miRNAs are a class of small, ncRNA molecules predicted to post-transcriptionally regulate at least one third of human genes (Lewis et al 2005). Each miRNA can potentially target hundreds of genes and play key regulatory roles in a diverse range of pathways including development, differentiation and pathological processes such as neurodegeneration (Enciu et al 2011). The study of miRNA in ALS is at a very early stage. However, given the proposed role for TDP-43 in miRNA biogenesis and the recent discovery of a beneficial effect of miRNA-206 in the mutant SOD1 mouse model, this will be an interesting area of investigation for the future (Buratti et al 2010; Williams et al 2009)
