Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease.

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.

Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis.

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3ß/ß-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3ß. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

Cilia, Centrosomes and Skeletal Muscle.

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.

WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis.

WDR62 is a scaffold protein involved in centriole duplication and spindle assembly during mitosis. Mutations in WDR62 can cause primary microcephaly and premature ovarian insufficiency. We have generated a genetrap mouse model deficient in WDR62 and characterised the developmental effects of WDR62 deficiency during meiosis in the testis. We have found that WDR62 deficiency leads to centriole underduplication in the spermatocytes due to reduced or delayed CEP63 accumulation in the pericentriolar matrix. This resulted in prolonged metaphase that led to apoptosis. Round spermatids that inherited a pair of centrioles progressed through spermiogenesis, however, manchette removal was delayed in WDR62 deficient spermatids due to delayed Katanin p80 accumulation in the manchette, thus producing misshapen spermatid heads with elongated manchettes. In mice, WDR62 deficiency resembles oligoasthenoteratospermia, a common form of subfertility in men that is characterised by low sperm counts, poor motility and abnormal morphology. Therefore, proper WDR62 function is necessary for timely spermatogenesis and spermiogenesis during male reproduction.

TDP-43 Mutation Affects Stress Granule Dynamics in Differentiated NSC-34 Motoneuron-Like Cells.

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of motor neurons in the brain and spinal cord. Cytoplasmic inclusions of TDP-43 are frequently reported in motor neurons of ALS patients. TDP-43 has also been shown to associate with stress granules (SGs), a complex of proteins and mRNAs formed in response to stress stimuli that temporarily sequester mRNA translation. The effect of pathogenic TDP-43 mutations within glycine-rich regions (where the majority of ALS-causing TDP-43 mutations occur) on SG dynamics in motor neurons is poorly understood. To address this issue, we generated murine NSC-34 cell lines that stably over-express wild type TDP-43 (TDP-43 W T ) or mutant forms (ALS-causing TDP-43 mutations TDP-43 A315T or TDP-43 M337V). We then differentiated these NSC-34 lines into motoneuron-like cells and evaluated SG formation and disassembly kinetics in response to oxidative or osmotic stress treatment. Wild type and mutant TDP-43 appeared to be largely retained in the nucleus following exposure to arsenite-induced oxidative stress. Upon arsenite removal, mutant TDP-43 clearly accumulated within HuR positive SGs in the cytoplasm, whereas TDP-43 W T remained mostly within the nucleus. 24 h following arsenite removal, all SGs were disassembled in both wild type and mutant TDP-43 expressing cells. By contrast, we observed significant differences in the dynamics of mutant TDP-43 association with SGs in response to hyperosmotic stress. Specifically, in response to sorbitol treatment, TDP-43 W T remained in the nucleus, whereas mutant TDP-43 relocalized to HuR positive SGs in the cytoplasm following exposure to sorbitol stress, resulting in a significant increase in TDP-43 SG numbers. These SGs remained assembled for 24 h following removal of sorbitol. Our data reveal that under certain stress conditions the rates of SG formation and disassembly is modulated by TDP-43 mutations associated with ALS, and suggest that this may be an early event in the seeding of insoluble cytoplasmic inclusions observed in ALS.

Pathophysiological Significance of WDR62 and JNK Signaling in Human Diseases.

The c-Jun N-terminal kinase (JNK) is highly evolutionarily conserved and plays important roles in a broad range of physiological and pathological processes. The WD40-repeat protein 62 (WDR62) is a scaffold protein that recruits different components of the JNK signaling pathway to regulate several human diseases including neurological disorders, infertility, and tumorigenesis. Recent studies revealed that WDR62 regulates the process of neural stem cell mitosis and germ cell meiosis through JNK signaling. In this review we summarize the roles of WDR62 and JNK signaling in neuronal and non-neuronal contexts and discuss how JNK-dependent signaling regulates both processes. WDR62 is involved in various human disorders via JNK signaling regulation, and may represent a promising therapeutic strategy for the treatment of related diseases.

The Spindle-Associated Microcephaly Protein, WDR62, Is Required for Neurogenesis and Development of the Hippocampus.

Primary microcephaly genes (MCPH) are required for the embryonic expansion of the mammalian cerebral cortex. However, MCPH mutations may spare growth in other regions of the developing forebrain which reinforces context-dependent functions for distinct MCPH genes in neurodevelopment. Mutations in the MCPH2 gene, WD40-repeat protein 62 (WDR62), are causative of primary microcephaly and cortical malformations in humans. WDR62 is a spindle microtubule-associated phosphoprotein that is required for timely and oriented cell divisions. Recent studies in rodent models confirm that WDR62 loss or mutation causes thinning of the neocortex and disrupted proliferation of apical progenitors reinforcing critical requirements in the maintenance of radial glia. However, potential contributions for WDR62 in hippocampal development had not been previously defined. Using CRISPR/Cas9 gene editing, we generated mouse models with patient-derived non-synonymous missense mutations (WDR62V66M and WDR62R439H) and a null mutation (herein referred to as WDR62Stop) for comparison. We find that WDR62 deletion or mutation resulted in a significant reduction in the thickness of the hippocampal ventricular zone and the area of the dentate gyrus (DG). This was associated with the mitotic arrest and depletion of radial glia and intermediate progenitors in the ammonic neuroepithelium. As a consequence, we find that the number of mitotic dentate precursors in the migratory stream and granule neurons in the DG was reduced with WDR62 mutation. These findings reveal that WDR62 is required for neurogenesis and the growth of the hippocampus during embryonic development.

Centrosome Reduction Promotes Terminal Differentiation of Human Cardiomyocytes.

Centrosome reduction and redistribution of pericentriolar material (PCM) coincides with cardiomyocyte transitions to a post-mitotic and matured state. However, it is unclear whether centrosome changes are a cause or consequence of terminal differentiation. We validated that centrosomes were intact and functional in proliferative human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), consistent with their immature phenotype. We generated acentrosomal hPSC-CMs, through pharmacological inhibition of centriole duplication, and showed that centrosome loss was sufficient to promote post-mitotic transitions and aspects of cardiomyocyte maturation. As Hippo kinases are activated during post-natal cardiac maturation, we pharmacologically activated the Hippo pathway using C19, which was sufficient to trigger centrosome disassembly and relocalization of PCM components to perinuclear membranes. This was due to specific activation of Hippo kinases, as direct inhibition of YAP-TEAD interactions with verteporfin had no effect on centrosome organization. This suggests that Hippo kinase-centrosome remodeling may play a direct role in cardiac maturation.

Elevated levels of Drosophila Wdr62 promote glial cell growth and proliferation through AURKA signalling to AKT and MYC.

WD40-Repeat Protein 62 (WDR62) is required to maintain neural and glial cell populations during embryonic brain growth. Although elevated expression of WDR62 is frequently associated with several tumour types, potential effects of excess WDR62 on proliferative growth remain undefined. Here, we demonstrate that glia specific overexpression of WDR62 in Drosophila larval brains resulted in increased cell size, over-proliferation and increased brain volume, without overt disruption of tissue organization. We further demonstrate WDR62 promoted over-proliferation and brain overgrowth by activating AURKA and pAKT signalling to increase MYC function in glial cells. Together these data suggest WDR62 normally functions in the glial lineage to activate oncogenic signalling networks, promoting proliferation and brain overgrowth.

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

Pathogenic E2K mutation of doublecortin X (DCX) alters microtubule stabilisation and actin filament association.

Mutations of the microtubule (MT)-associated protein Doublecortin X (DCX) gene disrupt cortical layering in brain development. Whilst many of these pathogenic DCX mutations are within the doublecortin domains (DC1 and DC2) that mediate direct DCX-MT association, a pathogenic mutation DCX E2K that causes cognitive impairment and pachygyria in human patients lies within the regulatory DCX N-terminus (DCX-N) preceding the DC1 domain. Here, we characterise the impact of DCX E2K on cytoskeletal association and regulation in neuronal cells. We show that the DCX E2K mutant protein retains the ability to interact with and bundle MTs, but these MTs show a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchange rates. Furthermore, we showed increased association of DCX E2K mutant with the actin filament (F-ACT) network. These results highlight the importance of the N-terminus of DCX in regulating association and co-ordination of MT and F-ACT networks.

Quantitative proteomic analyses of dynamic signalling events in cortical neurons undergoing excitotoxic cell death.

Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death. Our data, available in ProteomeXchange with identifier PXD008353, identified over 100 changed proteins exhibiting significant alterations in abundance and/or phosphorylation levels at different time points (5-240 min) in neurons after glutamate overstimulation. Bioinformatic analyses predicted that many of them are components of signalling networks directing defective neuronal morphology and functions. Among them, the well-known neuronal survival regulators including mitogen-activated protein kinases Erk1/2, glycogen synthase kinase 3 (GSK3) and microtubule-associated protein (Tau), were selected for validation by biochemical approaches, which confirmed the findings of the proteomic analysis. Bioinformatic analysis predicted Protein Kinase B (Akt), c-Jun kinase (JNK), cyclin-dependent protein kinase 5 (Cdk5), MAP kinase kinase (MEK), Casein kinase 2 (CK2), Rho-activated protein kinase (Rock) and Serum/glucocorticoid-regulated kinase 1 (SGK1) as the potential upstream kinases phosphorylating some of the changed proteins. Further biochemical investigation confirmed the predictions of sustained changes of the activation states of neuronal Akt and CK2 in excitotoxicity. Thus, future investigation to define the signalling pathways directing the dynamic alterations in abundance and phosphorylation of the identified changed neuronal proteins will help elucidate the molecular mechanism of neuronal death in excitotoxicity.

Doublecortin X (DCX) serine 28 phosphorylation is a regulatory switch, modulating association of DCX with microtubules and actin filaments.

Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275-366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1-44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and ß-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation.

MEKK3 coordinates with FBW7 to regulate WDR62 stability and neurogenesis.

Mutations of WD repeat domain 62 (WDR62) lead to autosomal recessive primary microcephaly (MCPH), and down-regulation of WDR62 expression causes the loss of neural progenitor cells (NPCs). However, how WDR62 is regulated and hence controls neurogenesis and brain size remains elusive. Here, we demonstrate that mitogen-activated protein kinase kinase kinase 3 (MEKK3) forms a complex with WDR62 to promote c-Jun N-terminal kinase (JNK) signaling synergistically in the control of neurogenesis. The deletion of Mekk3, Wdr62, or Jnk1 resulted in phenocopied defects, including premature NPC differentiation. We further showed that WDR62 protein is positively regulated by MEKK3 and JNK1 in the developing brain and that the defects of wdr62 deficiency can be rescued by the transgenic expression of JNK1. Meanwhile, WDR62 is also negatively regulated by T1053 phosphorylation, leading to the recruitment of F-box and WD repeat domain-containing protein 7 (FBW7) and proteasomal degradation. Our findings demonstrate that the coordinated reciprocal and bidirectional regulation among MEKK3, FBW7, WDR62, and JNK1, is required for fine-tuned JNK signaling for the control of balanced NPC self-renewal and differentiation during cortical development.

Flavonols and Flavones - Protecting Against Myocardial Ischemia/Reperfusion Injury by Targeting Protein Kinases.

In acute myocardial infarction (AMI), the first line of treatment is to rapidly restore blood flow to the ischemic myocardium to limit infarct size. It is now well established that though clearly beneficial, the positive outcomes of this intervention are limited by injury in response to the reperfusion itself in addition to the prior ischemia. This process is described as reperfusion injury and is considered to contribute to the arrhythmias, microvascular dysfunction and impaired cardiac contractility that is observed even after the restoration of coronary blood flow. Thus an important, currently unmet, therapeutic challenge is to address the outcomes of this reperfusion injury. In this article, we review the evidence that flavonols and flavones may prove useful in preserving cardiac function after ischemia and reperfusion and consider the possible mechanisms, in particular, the inhibition of kinases, by which they may exert protection.

Protection against reperfusion injury by 3',4'-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2.

BACKGROUND: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. METHODS: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10µM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. RESULTS: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. CONCLUSIONS: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.

Dynamic microtubule association of Doublecortin X (DCX) is regulated by its C-terminus.

Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association.

Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth.

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

A beacon of hope in stroke therapy-Blockade of pathologically activated cellular events in excitotoxic neuronal death as potential neuroprotective strategies.

Excitotoxicity, a pathological process caused by over-stimulation of ionotropic glutamate receptors, is a major cause of neuronal loss in acute and chronic neurological conditions such as ischaemic stroke, Alzheimer's and Huntington's diseases. Effective neuroprotective drugs to reduce excitotoxic neuronal loss in patients suffering from these neurological conditions are urgently needed. One avenue to achieve this goal is to clearly define the intracellular events mediating the neurotoxic signals originating from the over-stimulated glutamate receptors in neurons. In this review, we first focus on the key cellular events directing neuronal death but not involved in normal physiological processes in the neurotoxic signalling pathways. These events, referred to as pathologically activated events, are potential targets for the development of neuroprotectant therapeutics. Inhibitors blocking some of the known pathologically activated cellular events have been proven to be effective in reducing stroke-induced brain damage in animal models. Notable examples are inhibitors suppressing the ion channel activity of neurotoxic glutamate receptors and those disrupting interactions of specific cellular proteins occurring only in neurons undergoing excitotoxic cell death. Among them, Tat-NR2B9c and memantine are clinically effective in reducing brain damage caused by some acute and chronic neurological conditions. Our second focus is evaluation of the suitability of the other inhibitors for use as neuroprotective therapeutics. We also discuss the experimental approaches suitable for bridging our knowledge gap in our current understanding of the excitotoxic signalling mechanism in neurons and discovery of new pathologically activated cellular events as potential targets for neuroprotection.

Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation.

Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.