Our study provides a mechanism by which mutations in the human ASPM
gene could result in a developmental reduction of brain size, and supports the hypothesis that the MCPH phenotype arises due to defective NPC division. By examining the consequences of siRNA mediated knockdown of spindle pole associated ASPM expression and the phenotypes seen following expression of fusion proteins derived from the ASPM C
-terminus we have identified critical roles for human ASPM in spindle microtubule organisation, spindle positioning and in the regulation of cytokinesis even in a non-neural cell type. Similar phenotypes have been previously reported in Drosophila asp
]. We suggest that ASPM is a reasonably stable protein and that nuclear matrix associated ASPM is less open to degradation than ASPM after nuclear envelope breakdown. ASPM was recently confirmed as a gene involved in the regulation of mitosis in human cells as part of the substantive MitoCheck integrated research project ([42
). As an element of this high-throughput whole genome RNAi screen, automated live cell image analysis of ASPM knockdown identified similar phenotypes to those identified in our study. Although mitotic spindle position was not a phenotype for which analytical parameters were specifically established, other mitotic phenotypes that could lead to a decrease in cell division were identified. Metaphase delay was robustly observed with further phenotypes of mitotic delay, problems with metaphase alignment, cell death and poly-lobed nuclei identified with one of two siRNAs utilised.
We have previously shown that Aspm
is preferentially expressed during cerebral cortical neurogenesis in the mouse brain [5
]. The time of maximal Aspm
expression in the neuroepithelium corresponds to the period of proliferative cell division, and the subsequent down-regulation of Aspm
expression is concomitant with a switch to asymmetrical cell division [35
]. We have now demonstrated that inhibition of ASPM function by siRNA causes a highly penetrant loss of precision in the placement of the mitotic spindle in dividing U2OS cells, resulting in an alteration of division mode from the symmetrical to the asymmetrical plane. This implies that in U2OS cells symmetrical cell division in a plane that is perpendicular to the substrate is an active process that requires functional ASPM, rather than a simple default pathway. Our data therefore demonstrates that the requirement for ASPM in the maintenance of symmetrical divisions is not limited to specialised NPCs as previously assumed, but that it plays a general role in mitotic cells.
A logical consequence of this hypothesis is that the normal development of the whole human body should be affected by ASPM loss of function. Indeed in Drosophila
loss of asp function results in larval lethality [25
]. Paradoxically however, the only known phenotype in individuals with homozygous mutations in ASPM
is MCPH [5
]. Mutations in ASPM
are scattered throughout the gene, yet they result in a single clinical MCPH phenotype. Our immunoblot data of ASPM expression in fibroblast lysates from an MCPH patient carrying the IVS25+1G > T homozygous mutation established that the MCPH phenotype in this patient did not result from complete ASPM loss and confirmed a previous report that ASPM
mutation does not instigate the NMD pathway [8
]. Partly functional ASPM proteins may therefore be expressed in the cells of MCPH individuals, and we hypothesised that ASPM
mutations caused protein truncations and that the MCPH phenotype resulted from the common loss of a C-
terminal functional domain that led to partial loss of ASPM function. Surprisingly, our data from patient cells demonstrates that the MCPH phenotype can arise from a cryptic splicing event that removes only nine nucleotides of intragenic sequence within the C
-terminal region of ASPM and results in a reduction in ASPM localisation to the spindle poles. The minimal IVS25+1G > T mutation therefore causes a decrease in the efficiency of ASPM spindle pole localisation without an associated decrease in overall ASPM protein levels or a major increase in gross mitotic abnormalities in patient fibroblasts. We propose that expression of this subtly impaired mutant ASPM protein is sufficient to induce small deviations in the precision of mitotic spindle positioning in symmetrically dividing cells. This contrasts with the severe mitotic phenotypes seen in cultured cells following more profound disruption of ASPM function by siRNA knockdown of ASPM spindle pole expression, or expression of dominant negative ASPM C
The existence of individuals homozygous for mutations in ASPM
who exhibit a small brain but who are otherwise grossly normal leads us to infer that either (a) a functional compensatory mechanism exists in somatic cells or (b) neurogenic cell divisions are sufficiently different to the majority of somatic cell divisions to be profoundly and differentially affected by a subtle perturbation of ASPM function. The reason for this may lie in the unique morphology of the cells undergoing division in the neuroepithelium during cortical expansion [35
]. In vertebrates, apical NPC are apicobasally elongated to a remarkable extent and possess a small apical surface. To execute accurate symmetrical divisions in such cells would require extremely precise cleavage along the apicobasal axis [43
]. Small deviations in spindle position leading to cleavage plane reorientation would result in a transition from symmetrical to asymmetrical division. In the majority of somatic cells such deviations could be tolerated due to their relatively large apical and basal surfaces. However in NPC such deviation may be sufficient to drive a decrease in the number of cells successfully completing symmetrical cell division. This would impair the expansion of the progenitor pool that normally occurs at early stages of cortical development. As a consequence, the NPC pool would be insufficient to produce the number of neurones required for a normal sized brain.
What then is the function of ASPM during the later stages of mitosis? We observed an ASPM localisation at the minus ends of central spindle microtubules during anaphase and at the centre of the midbody during telophase and cytokinesis. Microtubules of the mitotic apparatus are a critical contributor to cleavage furrow positioning. Both astral microtubules and overlapping equatorial MTs in the central spindle have been implicated as playing a significant role in this process [44
]. We therefore see a number of possibilities for ASPM function during cytokinesis. ASPM might directly contribute to the organisation of midzone microtubules with the cytokinesis defects seen after ASPM functional inhibition arising secondary to central spindle disorganisation. Alternatively, its presence at the centrosome might influence the function of astral microtubules. It also seems possible that ASPM might participate more directly in the coupling of spindle microtubule function to cortical events during furrowing. For example, ASPM might be able to influence the local activity of myosins through its interactions with EF-hand Ca2+ binding factors such as calmodulin, or to influence signalling events during cytokinesis through interactions with binding partners such as citron kinase [38
In conclusion, a major finding of our study is that ASPM plays a role in cell division, not just those in NPCs, but in other cell types. We propose that MCPH5
patients have enough residual ASPM activity to successfully complete functionally symmetrical cell divisions in all tissues except the developing brain, where extremely unusual morphological constraints result in a specific defect in cortical neurogenic mitosis in response to imprecise spindle position. In this we echo the arguments presented by Fish et al
., in an authoritative commentary on NPC division [43
]. We have also identified a three amino acid sequence in the C
-terminal domain of ASPM in an MCPH patient reduces localisation of ASPM to the PCM implying that this region of ASPM mediates an important function in NPCs. Future studies will be aimed at defining whether this region of ASPM mediates an important interaction with a novel binding partner.