Date of Award

2014

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Professor Jochen H.M. Prehn

Second Supervisor

Dr Marion Hogg

Funder/Sponsor

Science Foundation Ireland

Keywords

Amyotrophic Lateral Sclerosis, Caspase

Abstract

Caspases are a major contributor to the apoptotic pathway. We intended to investigate the role of caspases, namely caspase-6, which has not been fully characterized in Amyotrophic Lateral Sclerosis (ALS). We utilized quantitative PCR (qPCR) to determine caspase-6 mRNA expression in nervous tissue from neocortex as well as ntg SOD1G93A ; caspase-6 -/- gastrocnemius muscle (pnd 35), ntg SOD1G93A ; caspase-6 +/+ gastrocnemius muscle (pnd 35), and compared this with purified primary motorneurons, microglia-enriched and astrocyteenriched cultures from primary E12 spinal cord tissue. No significant differences in caspase- 6 mRNA expression were detected in the sample types examined compared to nervous tissue.

We decided to investigate if caspase-6 protein levels were altered in a transgenic mouse model of ALS. We measured pro caspase-6 levels in tg and ntg SOD1G93A lumbar spinal cord at pnd 70, pnd 120, and pnd endstage (150-160 days). We found that pro caspase-6 showed a tendency towards an increase with the progression of ALS, yet this was is not at statistically significant levels. To further elucidate the role of caspase-6 during ALS disease progression, we developed a cross-breeding program to produce a SOD1G93A mouse colony deficient for caspase-6. The loss of motor function is a phenotypic indicator of ALS disease onset and progression in transgenic SOD1G93A mice. Motor function and behavioural analysis paradigms were used to assess the effect of caspase-6 deletion on functional defects and the lifespan of SOD1G93A ; caspase-6 colony. Surprisingly, we found that caspase-6 deficiency does not protect limb function, endurance, and motility in SOD1G93A mice and has no effect on body weight. We recorded the lifespan of SOD1G93A ; caspase-6 -/- mice to examine the effect of caspase-6 deletion during disease progression. Non-parametric analysis revealed that genetic deletion of caspase-6 did not increase survival in tg SOD1G93A ; caspase-6 -/- mice compared to tg SOD1G93A ; caspase-6 +/+ littermates, in fact it significantly decreased survival.

It has been hypothesized that caspase-6 may have a role in the degeneration in skeletal muscle that occurs prior to the onset of motorneuron degeneration in the spinal cord (Graham et al. 2006). We wanted to determine if motor endplates from hindlimb skeletal muscle showed any morphological changes in caspase-6 deficient mice compared to ntg SOD1G93A mice. Our findings showed no obvious signs of variation. We also compared weight and morphology of gastrocnemius muscle in SOD1G93A mice and showed that deletion of caspase-6 does not affect muscular atrophy in this mouse model for ALS.

Our initial hypothesis was that deletion of caspase-6 would protect neurons against cell death and therefore improve the phenotype in the SOD1G93A ALS mouse model. However we found the opposite were true and that deletion of caspase-6 enhanced disease progression and decreased lifespan. We hypothesize that caspase-6 is protective during ALS pathogenesis as the degenerative phenotype is worse when caspase-6 is removed, however further work is required to fully characterise the role of caspase-6.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

2.34 MB

Comments

This thesis is submitted to the Royal College of Surgeons in Ireland for the Degree of Master of Science (MSc) 2014.

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