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Title: Investigating RNA editing in the pathogenesis of amyotrophic lateral sclerosis
Author: Alsomali, Khayria
ISNI:       0000 0004 5357 8906
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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The susceptibility of MNs to injury in amyotrophic lateral sclerosis (ALS) may result from excitotoxicity and subsequent dysregulation of intracellular calcium homeostasis. Post-transcriptional editing of the AMPA and Kainate glutamate receptor (GLUR) subunit(s) may alter the calcium ion permeability of the receptor pore, leading to neurodegeneration. The aims of the study are (i) to investigate GLUR2, GLUR5 and GLUR6 RNA editing at Q/R site and its editing enzyme ADAR2 and ADAR3 in ALS/C9ORF72-positive and ALS/C9OR72-negative compared to non neurological controls, and (ii) to determine the aberrant expression of EAAT2 transcripts in the astrocytes of the spinal cord of ALS cases and controls. (iii) To characterise p-TDP43, p62, GLUR2, ADAR2, ADAR3 and GFAP and EAAT2 proteins in motor neurons of ALS/C9ORF72-postive and ALS/C9ORF72-negative cases and controls. (iv) To develop new methodology to look at different levels of the mRNA expression in GLUR2, GLUR5, GLUR6, ADAR2, ADAR3 and EAAT2 that might exist in ALS. RNA was extracted from MNs and cDNA was prepared followed by amplification of cDNA by PCR that was used to generate amplified GLUR2, GLUR5 and GLUR6. These products were digested to produce cuts in the internal site of GLUR mRNA and differentiate edited from non-edited GLUR by analysis using agarose gel electrophoresis and the Agilent Bioanalyser. Independently the mRNA expression of ADAR2, ADAR3 and EAAT2 alternative transcripts of normal and aberrant exon 4 and 9 were quantified by qPCR. p-TDP-43, p62, GLUR2, ADAR2, ADAR3 and GFAP and EAAT2 protein levels were assessed by immunohistochemistry. The mRNA expression of GLUR2, GLUR5, GLUR6, ADAR2, ADAR3 and EAAT2 was assessed by next generation sequencing It was demonstrated that GLUR2 was fully edited in the MNs of ALS/C9ORF72- positive, ALS/C9ORF72-negative cases and controls. GLUR5 was 39% edited in MNs of ALS/C9ORF72-positive, 57% edited in ALS/C9ORF72-negative cases and 30% edited in controls. GLUR6 editing in MNs was 86% editing in the ALS/C9ORF72- positive and 77% editing in ALS/C9ORF72-negative cases compared to 72% editing in controls. An elevated mRNA expression in EAAT2 aberrant and normal transcripts of exon 4 and exon 9 was shown, but did not reach tatistical significance. ADAR2 immunoreactivity was associated with the nuclei of MNs, in contrast to ADAR3 which was exclusively associated with the neuronal cytoplasm. No significant differences in the pattern or distribution of immunoreactivity was detected across all groups. Phosphorylated TDP-43+ and p62+ skein-like bodies and compact inclusions were significantly elevated in both ALS/C9ORF72-positive cases and ALS/C9ORF72- negative cases, compared to control subjects which exhibited neither p-TDP-43 nor p62 expression. No significant difference in the pattern of immunoreactivity of EAAT2 in ALS groups and controls. In conclusion, full editing of GLUR2 in dissected motor neurons isolated by LCM was confirmed in ALS/C9ORF72-positive and ALS/C9ORF72-negative and control individuals. Whereas partial editing has been identified in GLUR5 and GLUR6 receptors. The mRNA and protein expression of ADAR2 and ADAR3 on spinal cord have shown similar levels between tested groups. No significant difference in the mRNA and protein expression of EAAT2. MNs in ALS cases, both with and without C9ORF72 expansion expressed pathogenic inclusions of phosphorylated TDP-43 and p62, which are a hallmark for ALS. ADAR2 and ADAR3 have shown no differences in ALS groups compared to controls.
Supervisor: Heath, Paul ; Simpson, Julie Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available