Investigating the Nuclear Function of the C9orf72 Protein in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disorder where
death of the upper and lower motor neurons causes progressive muscle wasting
and paralysis. Currently, the approved treatments for ALS provide only a small
therapeutic benefit to patients. A GGGGCC repeat expansion mutation that lies
within the C9orf72 gene is causal of approximately 40% of the inherited form of
ALS. The mutation has been demonstrated to cause haploinsufficiency of the
C9orf72 protein and this loss of function is hypothesised to contribute to
pathogenesis in C9orf72-ALS. Until recently, little was known about the protein
function of C9orf72. We performed a yeast two-hybrid screen to investigate the
C9orf72 interactome which identified binding between C9orf72 and coilin. Coilin is
the major protein component of nuclear suborganelles, Cajal bodies, which are
responsible for processing snRNPs that form the spliceosome. C9orf72-ALS
patients exhibit splicing defects which correlate with disease severity. We therefore
hypothesised that loss of the C9orf72-coilin interaction leads to dysregulation of
Cajal bodies and splicing in C9orf72-ALS.
The aims of this project were to characterise the C9orf72-coilin interaction and
investigate the effect of C9orf72 on Cajal bodies and splicing. C9orf72 was found to
interact with coilin directly in vitro and within cells, suggesting the interaction has
biological importance. Depletion of C9orf72 in HEK293 cells led to an increase in
the number of Cajal bodies, and a subtle defect in splicing. In contrast, C9orf72
overexpression decreased the number of Cajal bodies, but had little effect on
splicing. Interestingly, investigation of Cajal bodies in C9orf72-ALS patient
iAstrocytes found patients exhibited higher numbers of Cajal bodies in comparison
to healthy controls.
The work included in this thesis uncovered an interaction between C9orf72 and
coilin and that depletion of C9orf72 can lead to abnormal numbers of Cajal bodies.
The results support evidence for a novel, uncharacterised role for C9orf72 in the
nucleus which may be linked with snRNP processing, Cajal bodies and splicing.
Investigation into the function of nuclear C9orf72 will lead to greater understanding
of the consequences of C9orf72 haploinsufficiency in ALS and may lead to
development of better drug treatments for patients.