Abnormal Spindle-like Microcephaly-associated Research Articles

ASPM promotes migration and invasion of anaplastic thyroid carcinoma by stabilizing KIF11.

Abnormal spindle-like microcephaly-associated (ASPM) protein is crucial to the mitotic spindle function during cell replication and tumor progression in multiple tumor types. However, the effect of ASPM in anaplastic thyroid carcinoma (ATC) has not yet been understood. The present study is to elucidate the function of ASPM in the migration and invasion of ATC. ASPM expression is incrementally upregulated in ATC tissues and cell lines. Knockout (KO) of ASPM pronouncedly attenuates the migration and invasion of ATC cells. ASPM KO significantly reduces the transcript levels of Vimentin, N-cadherin, and Snail and increases E-cadherin and Occludin, thereby inhibiting epithelial-to-mesenchymal transition (EMT). Mechanistically, ASPM regulates the movement of ATC cells by inhibiting the ubiquitin degradation of KIF11 and thus stabilizing it via direct binding to it. Moreover, xenograft tumors in nude mice proved that KO of ASPM could ameliorate tumorigenesis and tumor growth accompanied by a decreased protein expression of KIF11 and an inhibition of EMT. In conclusion, ASPM is a potentially useful therapeutic target for ATC. Our results also reveal a novel mechanism by which ASPM inhibits the ubiquitin process in KIF11.

MICROCEPHALY: A Developmental Disorder

Microcephaly is a result of abnormal in utero development resulting in an unusually small head size. The word comes from a New Latin word, termed as microcephalia, and from two Ancient Greek terminologies that translates into small and head. Back in Ancient Roman times, individuals that were known to carry small heads in comparison to a normal sized human head were all showcased as a public display, whereas people with microcephaly have been occasionally traded to multiple freak shows back in European and North American regions in the nineteenth and twentieth centuries, in which they were regarded as pinheads or freaks of the nature while a lot among them were showcased as a completely different species and were referred to as the lost connection in the evolution process. Growth limitation impacts the Central Nervous System (CNS) and brain in the more serious forms of cases, which might also result in significant perpetual cognitive deficits if the infant manages to survive. Microcephaly is caused by a number of variables, including chromosomal abnormalities as well as other genetic conditions, infections during pregnancy, for instance; rubella, toxoplasmosis, and prenatal exposure to dangerous toxicants.
 Although drastically deficient cognitive growth is prevalent, problems with motor control processes not showing up until much later in life. Most negatively impacted infants have severe neurological abnormalities and sometimes seizures, as well. Motor function and verbal advancement may also be deferred while hyperactivity and intellectual disability are both prevalent, however to varying degrees. Convulsions are also possible with variations in motor ability; from clumsiness to spastic quadriplegia in some people. The majority of cases of microcephaly are caused by genetic variations. On the one hand, linkage has been discovered between autism, gene duplications, and macrocephaly. On the contrary, a link has been discovered between schizophrenia, gene removals, and microcephaly. Several genotypes, termed as "MCPH" genes, after the gene microcephalin (MCPH1), because of their involvement in cranial capacity and primary microcephaly neuropathies when mutations occur. WDR62 (MCPH2), ASPM (MCPH5), CDK5RAP2 (MCPH3), CENPJ (MCPH6), KNL1 (MCPH4), STIL (MCPH7), CEP152 (MCPH9), ZNF335 (MCPH10), CEP135 (MCPH8), CDK6 (MCPH12), and PHC1 (MCPH11) are among the other genes related to microcephalin gene. Furthermore, a link has been found among repeated genetic variations in recognised genes, including MCPH1 and CDK5RAP2 with structural variations of the brain as examined by Magnetic Resonance Imaging (MRI). The Centres for Disease Control and Prevention and the International Society for Infectious Diseases have linked the expansion of vector-borne Zika virus to an increase in genetically inherited microcephaly. Zika can be transmitted from a pregnant woman to her foetus.
 Being caused by a reduction in cerebral cortex, and it can occur throughout embryonic and foetal growth phases because of inadequate neural stem cell advancement, impeded neurogenesis, or decrease of neural stem cells. Many genes needed for standard neural development have been discovered through studies in animal models such as rodents. The genes associated with the Notch pathway, for instance, govern stem cell advancement and neurogenesis. Genetic variations induced in mouse models in an experimental setting can induce microcephaly that in comparison is similar to that of the human beings. Abnormal Spindle-like Microcephaly-Associated (ASPM) gene abnormalities have been linked to human microcephaly, and a knockout-model of a ferret with extreme microcephaly has now been designed. Furthermore, viruses like Zika virus and Cytomegalovirus (CMV) have been found to afflict and destroy the brain's primary stem cells and radial glial cells, resulting in the destruction of daughter neurons.
 A comprehensive physical and history assessments are carried out on patients with microcephaly. Neuroimaging, metabolic evaluation, and genetic examination should be taken into consideration in cases of deteriorating microcephaly. Neuroimaging with Magnetic Resonance Imaging (MRI) is mostly utilized as the very first diagnostic analysis in children suffering from microcephaly. Genetic screening is frequently the very next process after imaging techniques. Microcephaly is a long-term condition with no specific treatment available.

Loss of abnormal spindle-like, microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15–20 weeks, 40–50 weeks and 70–80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10–15 weeks, 40–50 weeks and 70–80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging.

A Two-Base Pair Deletion in IQ Repeats in ASPM Underlies Microcephaly in a Pakistani Family.

Aims: Autosomal recessive primary microcephaly (MCPH) is a clinically rare and genetically highly heterogeneous developmental disorder. Biallelic variants in the abnormal spindle-like microcephaly-associated (ASPM) gene account for 40% to 68% of all MCPH cases. This study was designed to elucidate the genetic basis of MCPH in an extended family. To highlight recurrent mutations useful in implementing genetic testing programs, we further aimed to carry out a descriptive review of the reported ASPM mutations. Materials and Methods: A large inbred kindred with seven affected members was investigated, and detailed clinical and behavioral assessments were carried out. Single nucleotide polymorphism (SNP)-based homozygosity mapping and exome sequencing were performed. Results: Affected individuals had characteristic features, including small head, receding forehead, mild to moderate intellectual disability, developmental delay, short stature, apraxia, and behavioral anomalies. We mapped the disease gene locus and detected a rare frameshift deletion c.6854_6855del (p.(Leu2285GlnfsTer32)) in exon 18 of ASPM. A total of 215 mutations in ASPM have been reported in at least 453 families, nearly 50% of which are of Pakistani origin. These mutations can be classified as recurrent, founder or private in Pakistani and other populations. Conclusion: SNP-based homozygosity mapping and exome sequencing are essential in delineating the genetically distinct microcephaly types. The highlighted recurrent mutations in ASPM could be useful in implementing genetic testing programs for MCPH.

ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability

SummaryDNA double-strand break (DSB) repair by homologous recombination (HR) is essential for ensuring genome stability. Abnormal spindle-like microcephaly-associated (ASPM) gene encodes a spindle protein that is commonly implicated in primary microcephaly. We found that ASPM is recruited to sites of DNA damage in a PARP2-dependent manner. ASPM interacts with BRCA1 and its E3 ligase HERC2, preventing HERC2 from accessing to BRCA1 and ensuring BRCA1 stability. Inhibition of ASPM expression promotes HERC2-mediated BRCA1 degradation, compromises HR repair efficiency and chromosome stability, and sensitizes cancer cells to ionizing radiation. Moreover, we observed a synergistic effect between ASPM and PARP inhibition in killing cancer cells. This research has uncovered a novel function for ASPM in facilitating HR-mediated repair of DSBs by ensuring BRCA1 stability. ASPM might constitute a promising target for synthetic lethality-based cancer therapy.

Overexpression of abnormal spindle-like microcephaly-associated (ASPM) increases tumor aggressiveness and predicts poor outcome in patients with lung adenocarcinoma.

BackgroundCumulative evidence points to abnormal spindle-like microcephaly-associated (ASPM) protein being overexpressed in various cancers, and the aberrant expression of ASPM has been shown to promote cancer tumorigenicity and progression. However, its role and clinical significance in lung adenocarcinoma (LUAD) remains unclear. This study aimed to determine the expression patterns of ASPM and its clinical significance in LUAD.MethodsIn total, 4 original worldwide LUAD microarray mRNA expression datasets (N=1,116) with clinical and follow-up annotations were downloaded from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. The expression of ASPM protein in LUAD patients was detected by immunohistochemistry. Survival analysis and Cox regression analysis were used to examine the prognostic value of ASPM expression. Gene set enrichment analysis (GSEA) was performed to investigate the relationship between ASPM and LUAD.ResultsDataset analyses and immunohistochemistry revealed that ASPM expression was significantly higher in the LUAD tissues compared with normal lung tissues, especially in the advanced tumor stage. Additionally, overexpression of ASPM was significantly correlated with shorter overall survival (OS) and relapse-free survival (RFS) in LUAD. Univariate and multivariate Cox regression analyses revealed that the overexpression of ASPM was a potential independent predictor of poor OS and RFS. However, ASPM overexpression was not significantly associated with predicting OS in lung squamous cell carcinoma. GSEA analysis demonstrated that ASPM was significantly enriched in the cell cycle, DNA replication, homologous recombination, RNA degradation, mismatch repair, and p53 signaling pathways.ConclusionsThese findings demonstrate the important role of ASPM in the tumorigenesis and progression of LUAD.

A multi-mode Wnt- and stemness-regulatory module dictated by FOXM1 and ASPM isoform I in gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer mortality globally and a molecularly heterogeneous disease. Identifying the driver pathways in GC progression is crucial to improving the clinical outcome. Recent studies identified ASPM (abnormal spindle-like microcephaly-associated) and FOXM1 (Forkhead box protein M1) as novel Wnt and cancer stem cell (CSC) regulators; their pathogenetic roles and potential crosstalks in GC remain unclarified. The expression patterns of ASPM isoforms and FOXM1 were profiled in normal gastric epithelial and GC tissues. The functional roles of ASPM and FOXM1 in Wnt activity, cancer stemness and GC progression, and the underlying signaling processes were investigated. Approximately one third of GC cells upregulate the expression of ASPM isoform I (ASPMiI) in their cytoplasm; the tumors with a high ASPMiI positive score (≥ 10%) are associated with a poor prognosis of the patients. Mechanistically, the molecular interplay among FOXM1, ASPMiI and DVL3 was found to converge on β-catenin to control the Wnt activity and the stemness property of GC cells. This multi-mode Wnt-regulatory module serves to reinforce Wnt signals in CSCs by transcriptional regulation (FOXM1-ASPM), protein-protein interactions (ASPMiI-DVL3-β-catenin), and nuclear translocation (FOXM1-β-catenin). This study illuminates a novel Wnt- and stemness-regulatory mechanism in GC cells and identifies a novel subset of FOXM1highASPMiIhigh GC with potential to guide Wnt- and stemness-related diagnostics and therapies.

Abnormal spindle-like microcephaly-associated (ASPM) gene expression in posterior fossa brain tumors of childhood and adolescence.

In neurogenesis, ASPM (abnormal spindle-like microcephaly-associated) gene is expressed mainly in the ventricular zone of posterior fossa and is the major determinant in the cerebral cortex. Besides its role in embryonic development, ASPM overexpression promotes tumor growth, including central nervous system (CNS) tumors. This study aims to investigate ASPM expression levels in most frequent posterior fossa brain tumors of childhood and adolescence: medulloblastoma (MB), ependymoma (EPN), and astrocytoma (AS), correlating them with clinicopathological characteristics and tumor solid portion size. Quantitative reverse transcription (qRT-PCR) is used to quantify ASPM mRNA levels in 80 pre-treatment tumor samples: 28MB, 22 EPN, and 30 AS. The tumor solid portion size was determined by IOP-GRAACC Diagnostic Imaging Center. We correlated these findings with clinicopathological characteristics and tumor solid portion size. Our results demonstrated that ASPM gene was overexpressed in MB (p = 0.007) and EPN (p = 0.0260) samples. ASPM high expression was significantly associated to MB samples from patients with worse overall survival (p = 0.0123) and death due to disease progression (p = 0.0039). Interestingly, two patients with AS progressed toward higher grade showed ASPM overexpression (p = 0.0046). No correlation was found between the tumor solid portion size and ASPM expression levels in MB (p = 0.1154 and r = - 0.4825) and EPN (p = 0.1108 and r = - 0.3495) samples. Taking in account that ASPM gene has several functions to support cell proliferation, as mitotic defects and premature differentiation, we suggest that its overexpression, presumably, plays a critical role in disease progression of posterior fossa brain tumors of childhood and adolescence.

Dishevelled 1-Regulated Superpotent Cancer Stem Cells Mediate Wnt Heterogeneity and Tumor Progression in Hepatocellular Carcinoma.

SummaryVarious populations of cancer stem cells (CSCs) have been identified in hepatocellular carcinoma (HCC). Wnt signaling is variably activated in HCC and regulates CSCs and tumorigenesis. We explored cell-to-cell Wnt and stemness heterogeneity in HCC by labeling freshly isolated cancer cells with a Wnt-specific reporter, thereby identifying a small subset (0.4%–8.9%) of Wnt-activityhigh cells. Further cellular subset analysis identified a refined subset of Wnt-activityhighALDH1+EpCAM+ triple-positive (TP) cells as the most stem-like, phenotypically plastic, and tumorigenic among all putative CSC populations. These TP “superpotent CSCs” (spCSCs) specifically upregulate the expression of dishevelled 1 (DVL1) through the antagonism between abnormal spindle-like microcephaly-associated (ASPM) and the ubiquitin ligase complex Cullin-3/KLHL-12. Subsequent functional and molecular studies revealed the role of DVL1 in controlling spCSCs and their tumorigenic potential. These findings provide the mechanistic basis of the Wnt and stemness heterogeneity in HCC and highlight the important role of DVL1high spCSCs in tumor progression.

The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment‐resistant malignancy. The lack of pathway‐informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle‐like microcephaly‐associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform‐specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM‐iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM‐iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM‐iII is predominantly expressed in cell nuclei. Mechanistically, ASPM‐iI co‐localizes with disheveled‐2 and active β‐catenin as well as the stemness marker aldehyde dehydrogenase‐1 (ALDH‐1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM‐iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM‐iI and ASPM‐iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM‐iI was prognostically significant; it outperformed ALDH‐1 staining and clinico‐pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Overexpression of the ASPM gene is associated with aggressiveness and poor outcome in bladder cancer.

Abnormal spindle-like microcephaly-associated (ASPM) protein is essential for mitotic spindle function during cell replication. The present study aimed to evaluate the hypothesis that ASPM serves a critical role in cancer invasiveness and may act as a prognostic biomarker in bladder cancer. In total, 6 independent worldwide bladder cancer microarray mRNA expression datasets (n=1,355) with clinical and follow-up annotations were collected from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Reverse transcription-quantitative polymerase chain reaction analysis revealed that ASPM mRNA expression was higher in bladder cancer tissue compared with adjacent normal bladder mucosae in 10 paired human tissue samples (P=0.004). ASPM overexpression in human bladder cancer samples was consistent with the mRNA expression datasets from GEO and TCGA. Bioinformatics analysis indicated that ASPM mRNA expression was significantly associated with grade and tumor node metastasis (TNM) stage in bladder cancer, based on pooled GEO and TCGA datasets (P<0.05). Stratification analysis indicated that the clinical significance of ASPM was particularly pronounced in low-grade or papillary subtypes of bladder cancer. Individual Cox and pooled Kaplan-Meier analyses suggested that ASPM expression was significantly directly correlated with poor overall (OS) and progression-free survival (PFS) in bladder cancer. Multivariate and stratification analyses demonstrated that the prognostic significance of ASPM was evident in low-grade or papillary bladder cancers, yet not in high-grade or non-papillary subgroups. Increased expression of ASPM was associated with poor OS in muscle-invasive bladder cancer and with poor PFS in non-muscle-invasive bladder cancer (P<0.05). Bioinformatics analysis identified the top 11 ASPM-related genes on STRING-DB.org. The expression of the majority of these genes was associated with poor outcomes of bladder cancer with statistical significance. Gene set enrichment analysis indicated that the high expression of ASPM could enrich gene signatures involved in mitosis, differentiation and metastasis in bladder cancer. Further analysis of TCGA datasets indicated that increased ASPM expression was significantly associated with higher Gleason score, T stage, N stage and poor clinical outcome in prostate cancer. It was also significantly associated with late TNM stage and poor PFS in renal cell carcinoma. In summary, ASPM may serve as a novel prognostic biomarker for low-grade or papillary bladder cancer.

Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding, yet the evolutionary mechanisms driving cortical size and structure are unknown. While genes essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (“small head”, associated with reduced brain size and intellectual disability)1, studies of these genes in mice, whose smooth cortex is one thousand times smaller than that of humans, have provided limited insight. Mutations of abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by ≥50% in humans2–4, but have little effect in mice5–9, likely reflecting evolutionarily divergent functions of ASPM10,11. We used genome editing to create a germline knockout (KO) of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell (NPC) diversity12–14 than mice, and closer Aspm protein sequence homology to human. Aspm KO ferrets exhibit severe microcephaly (25–40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as in human patients3,4, suggesting loss of “cortical units”. The mutant ferret fetal cortex displays a massive premature displacement of ventricular radial glial cells (VRG) to the outer subventricular zone (OSVZ), where many resemble outer radial glia (ORG), an NPC subtype essentially absent in mice and implicated in cerebral cortical expansion in primates12–16. These data suggest an evolutionary mechanism whereby Aspm regulates cortical expansion by controlling the affinity of VRG for the ventricular surface, thus modulating the ratio of VRG, the most undifferentiated cell type, to ORG, a more differentiated progenitor.

Primary microcephaly caused by novel compound heterozygous mutations in ASPM

Autosomal recessive primary microcephaly (microcephaly primary hereditary, MCPH) is a genetically heterogeneous rare developmental disorder that is characterized by prenatal onset of abnormal brain growth, which leads to intellectual disability of variable severity. We report a 5-year-old male who presented with a severe form of primary microcephaly. Targeted panel sequencing revealed compound heterozygous truncating mutations of the abnormal spindle-like microcephaly-associated (ASPM) gene, which confirmed the MCPH5 diagnosis. A novel NM_018136.4: c.9742_9745del (p.Lys3248Serfs*13) deletion mutation was identified.

Longitudinal Diffusion Tensor Imaging Revealed Nerve Fiber Alterations in Aspm Mutated Microcephaly Model Mice

Autosomal recessive primary microcephaly-5 (MCPH5) is characterized by congenital microcephaly and is caused by the mutation in the abnormal spindle-like, microcephaly-associated (ASPM) gene. This study aimed to demonstrate a correlation between radiological and pathological analyses in evaluating postnatal brain development using MCPH5-model mice, ASPM ortholog (Aspm) knockout (KO) mice. In vivo MRI was performed at two time points (postnatal 3 weeks; P3W and P10W) and complementary histopathological analyses of brains were done at P5W and P13W. In the MRI analysis, Aspm KO mice showed significantly decreased brain sizes (average 8.6% difference) with larger ventricles (average 136.4% difference) at both time points. Voxel-based statistics showed that the fractional anisotropy (FA) values were significantly lower in Aspm KO mice in both the cortex and white matter at both time points. Developmental changes in the FA values were less remarkable in the Aspm KO mice, compared with the controls. Histometric analyses revealed that the ratios of the horizontal to the vertical neurites were significantly higher in cortical layers IV, V and VI, with a remarkable increase according to maturation at P13W in the control mice (average 12.7% difference between control and KO), whereas the ratio in layer VI decreased at P13W in the KO mice. The myelin basic protein positive ratio in the white matter significantly decreased in Aspm KO mice at P5W. These results suggest that temporal FA changes are closely correlated with pathological findings such as abnormal neurite outgrowth and differentiation, which may be applicable for analyzing diseased human brain development.

Abstracts from the 3rd International Genomic Medicine Conference (3rd IGMC 2015) : Jeddah, Kingdom of Saudi Arabia. 30 November - 3 December 2015.

Abstracts from the 3rd International Genomic Medicine Conference (3rd IGMC 2015) : Jeddah, Kingdom of Saudi Arabia. 30 November - 3 December 2015.

Abnormal spindle-like microcephaly-associated (ASPM) mutations strongly disrupt neocortical structure but spare the hippocampus and long-term memory

Autosomal recessive primary microcephaly results from abnormal brain development linked to proliferation defects in neural progenitors. The most frequent form, caused by ASPM mutations, is usually defined by a reduced brain volume and is associated with intellectual disability. Although many ASPM cases have now been reported, structural brain abnormalities and their link with cognitive disabilities have rarely been investigated. In this study, we used high resolution T1-weighted magnetic resonance imaging in seven patients with ASPM mutations and 39 healthy age-matched controls to quantify regional volumes, thickness, surface area, gyrification index and white matter volumes of 30 cortical regions. We observed a consistent reduction of 50% or more in the volume and surface area of all cortical regions except for the hippocampus and surrounding medial temporal structures, which were significantly less reduced. Neuropsychologic assessment indicated significant impairments of cognitive abilities. However, these impairments were associated with normal mnesic abilities, in keeping with the relative preservation of the hippocampus and medial temporal structures. These results show that, contrary to current opinion, the cortical volume and surface area of patients with ASPM mutations is reduced depending on a regionally specific fashion and their cognitive profile reflects this heterogeneity. The precise characterization of the cortical map and cognitive abilities of patients with ASPM mutations should allow developing more focused reeducative interventions well-suited to their real abilities.

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.

ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy

Purpose: In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied.Materials and methods: Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA).Results: Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway.Conclusions: Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.

Abstract 5544: ASPM is a novel participant in DNA double-strand break repair and a potential target for radiotherapy

Abstract Comprehensive serach for human cellular response to ionizing radiation (IR) is indispensable strategy to understand the molecular basis under tumor radiotherapy. In a previous study using HiCEP, we found that ASPM (Abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene is selectively down-regulated by IR. Here we first demonstrate that down-regulation of ASPM by siRNA synergistically enhanced radiosensitivity in some immortalized human cell lines. Both constant-field gel electrophoresis and gamma-H2AX foci assay exhibited impaired DNA double-strand breaks (DSB) in the cells treated with the siRNA. Greater extent of chromosomal instability was also common in the treated cells than untreated control. Getting together, ASPM (MCPH5) is a novel participant in the DNA double-strand break repair pathways. Although roles of microcephalins (MCPH1, pericentrin, ATR) have recently been proposed on DNA damage repair and check point regulation, this is the first report on ASPM. In clinical aspects, ASPM would be a promising target for the future cancer treatment as well as a useful biomarker in the prognostic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5544.

Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells.

The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.