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RNAseq of laser captured spinal and oculomotor neurons in post mortem material of ALS patients

ABSTRACT: RNAseq of laser captured spinal and oculomotor neurons in post mortem material of ALS patients

PROVIDER: PRJNA473843 | ENA |

REPOSITORIES: ENA

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Project description:Amyotrophic lateral sclerosis (ALS) spares the ocular motor system. In this study, we tested the hypothesis that the oculomotor neurons are intrinsically protected in ALS. Using high-density cDNA microarrays, we examined the transcriptome of oculomotor nuclei and spinal cords in mice expressing a human mutant SOD1, the SOD1(G93A) ALS model, at 6 and 10 weeks of age. Comparison of gene expression profiles of these pre-symptomatic SOD1(G93A) mice showed a shift to a proapoptotic state in spinal cords, while the opposite was true in oculomotor nuclei. Seventeen members of the A, B, C and D Hox clusters increased in oculomotor nuclei from 6 to 10 weeks of age; 15 were downregulated in spinal cord. Although only the first 4 classes of a given Hox cluster (e.g., Hoxa1-4) are normally expressed in the developing hindbrain, we found differential expression of mostly the latter classes in both oculomotor nuclei and spinal cords. Also, semaphorin 3B was expressed at 28-fold greater levels in oculomotor nuclei and 61-fold less in spinal cords in 10-week old SOD1(G93A) mice compared to 6-week old mice. Semaphorins 3A and 3E were also differentially regulated. Comparison of gene expression profiles of control SOD1 mice of 6 and 10 weeks of age did not show these changes. Based on these results, we rejected our hypothesis and conclude that the oculomotor nuclei actively adapt to the ALS-inducing mutation.,Supported by NEI and ALSA. <br>Overall design<br>Oligonucleotide microarray studies using the Affymetrix system were conducted as described earlier (McMullen et al., 2004). Biotinylated cRNA was hybridized to Affymetrix Mouse Expression Set 430A GeneChips. Then, the microarrays were washed and stained with a streptavidin-bound marker, and scanned with a laser scanner. Resulting microarray data were analyzed with Affymetrix Microarray Suite 5.0 software. Only those genes with consistent absent/present calls in the three independent replicates per group were considered for further analyses. Comparisons were crossed such that each oculomotor nuclei sample was compared with each spinal cord sample at the corresponding age. The Affymetrix software uses the one-sided Wilcoxons signed rank test to estimate increase/no change/decrease difference calls and fold-changes for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes greater than 2-fold were considered significant, resulting in a conservative list of genes with changed expression levels.

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RNAseq of laser captured spinal and oculomotor neurons in post mortem material of ALS patients

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