Abstract
BackgroundMutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC).ResultsWe show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells.ConclusionsThese observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.
Highlights
Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation
We show that ASPM knockdown in U2OS osteosarcoma cells by Small interfering RNA (siRNA) alters the positioning of the mitotic spindle from parallel to the substrate to perpendicular to the substrate, effectively altering the division symmetry from symmetrical to asymmetrical
ASPM is a nuclear protein that relocates to the spindle pole matrix and central spindle MT minus ends during mitosis To determine the intracellular distribution of human ASPM, rabbit polyclonal antibodies were raised against ASPM-specific peptide sequences (Figure 1A) and used to screen a panel of cell lines by immunofluorescence
Summary
Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). Autosomal recessive primary microcephaly (MCPH) is a rare Mendelian disorder characterized by a congenital deficiency of foetal brain growth, affecting the neocortex. This results in the formation of a small but structurally normal brain and associated mental retardation but no other neurological defects [4,5]. The concept that MCPH is a primary disorder of neurogenic mitosis, the result of which is a reduction of cell number in the developing human brain, is an attractive one
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