Determining the role of ASPM in human brain size regulation.

Abstract

Autosomal recessive primary microcephaly (MCPH) is a disease consisting of a small brain and associated intellectual disability. The most common cause of MCPH is mutation of the abnormal spindle-like microtubule-associated gene (ASPM, formerly known as abnormal spindle-like microcephaly-associated gene) at the MCPH5 locus. This thesis studies the function of ASPM, mitotic alterations due to homozygous ASPM mutation and identifies ASPM C-terminal interactants. ASPM siRNA KD studies confirmed previously identified roles for ASPM in spindle pole focusing, spindle/cleavage furrow orientation, spindle assembly and cytokinesis. Roles for ASPM in maintaining nuclear and centrosome structure, centrosome biogenesis, mitotic progression, microtubule organisation, and abscission were identified as being microtubule associated. A novel role for ASPM in actin organisation was identified. In MCPH5 patient fibroblast cells carrying the homozygous nonsense ASPM mutation 3663delG, a low level of full length ASPM protein was observed suggesting transcriptional read-through was occurring. ASPM3663delG was mis-localised to the cytoplasm suggesting it has a reduced ability to either travel to the mitotic spindle pole or become associated at the pericentrosomal matrix. In the patient fibroblast cells carrying ASPM3663delG and ASPM9984+1T>G homozygous mutations microtubule defects caused a variety of aberrant mitotic phenotypes which induced an increase in the duration of mitosis. These defects included prometaphase (ASPM3663delG) or metaphase (ASPM9984+1G>T) arrest, spindle mis-orientation and abscission failure and a decrease in the incidence of proliferation. Therefore MCPH arises

from a combination of mitotic abnormalities rather than a single aberration, which together result in a reduction in neural progenitor cell number. It is proposed that the single MCPH5 small brain phenotype arising in patients carrying homozygous nonsense ASPM mutations may be the result of transcriptional read-through. ASPM C--terminus has been shown to be required for ASPM spindle pole localisation, microtubule organisation and cytokinesis. Microtubule actin cross-linking factor 1 (MACF1) was identified as a candidate ASPM interacting protein from a yeast-2 Hybrid screen of the ASPM C-terminus against a foetal brain cDNA library. The MACF1-ASPM association was validated by co-immunoprecipitation (co-IP). MACF1 was shown to be associated with microtubules during interphase and was dependent upon ASPM for this localisation. MACF1 mitotic localisation was consistent with ASPM localisation at the minus ends of spindle microtubules and pericentrosomal matrix from prometaphase to early telophase. In patient cells carrying an ASPM C-terminal mutation MACF1 protein levels were proportional to ASPM expression. The alteration in the ASPM-MACF1 association due to ASPM mutation or ASPM reduction may account for the changes in actin organisation, microtubule associated forces, spindle mis-orientation and abscission observed in this research study.