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Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic CIT missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it does not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lose cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupt polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Autosomal recessive primary microcephaly (MCPH) is a constellation of disorders that share significant brain size reduction and mild to moderate intellectual disability, which may be accompanied by a large variety of more invalidating clinical signs. Extensive neural progenitor cells (NPC) proliferation and differentiation are essential to determine brain final size. Accordingly, the 30 MCPH loci mapped so far (MCPH1-MCPH30) encode for proteins involved in microtubule and spindle organization, centriole biogenesis, nuclear envelope, DNA replication and repair, underscoring that a wide variety of cellular processes is required for sustaining NPC expansion during development. Current models propose that altered balance between symmetric and asymmetric division, as well as premature differentiation, are the main mechanisms leading to MCPH. Although studies of cellular alterations in microcephaly models have constantly shown the co-existence of high DNA damage and apoptosis levels, these mechanisms are less considered as primary factors. In this review we highlight how the molecular and cellular events produced by mutation of the majority of MCPH genes may converge on apoptotic death of NPCs and neurons, via TP53 activation. We propose that these mechanisms should be more carefully considered in the alterations of the sophisticated equilibrium between proliferation, differentiation and death produced by MCPH gene mutations. In consideration of the potential druggability of cell apoptotic pathways, a better understanding of their role in MCPH may significantly facilitate the development of translational approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Iegiani, Ferraro, Pallavicini and Di Cunto.)

Introduction: Medulloblastoma (MB), the most common malignant pediatric brain tumor, is currently treated with surgery followed by radiation and chemotherapy, which is accompanied by severe side effects, raising the need for innovative therapies. Disruption of the microcephaly-related gene Citron kinase (CITK) impairs the expansion of xenograft models as well as spontaneous MB arising in transgenic mice. No specific CITK inhibitors are available., Methods: Lestaurtinib, a Staurosporine derivative also known as CEP-701, inhibits CITK with IC50 of 90 nM. We therefore tested the biological effects of this molecule on different MB cell lines, as well as in vivo, injecting the drug in MBs arising in SmoA1 transgenic mice., Results: Similar to CITK knockdown, treatment of MB cells with 100 nM Lestaurtinib reduces phospho-INCENP levels at the midbody and leads to late cytokinesis failure. Moreover, Lestaurtinib impairs cell proliferation through CITK-sensitive mechanisms. These phenotypes are accompanied by accumulation of DNA double strand breaks, cell cycle block and TP53 superfamily activation in vitro and in vivo. Lestaurtinib treatment reduces tumor growth and increases mice survival., Discussion: Our data indicate that Lestaurtinib produces in MB cells poly-pharmacological effects extending beyond the inhibition of its validated targets, supporting the possibility of repositioning this drug for MB treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pallavicini, Iegiani, Parolisi, Ferraro, Garello, Bitonto, Terreno, Gai and Di Cunto.)

In the developing mouse forebrain, temporally distinct waves of oligodendrocyte progenitor cells (OPCs) arise from different germinal zones and eventually populate either dorsal or ventral regions, where they present as transcriptionally and functionally equivalent cells. Despite that, developmental heterogeneity influences adult OPC responses upon demyelination. Here we show that accumulation of DNA damage due to ablation of citron-kinase or cisplatin treatment cell-autonomously disrupts OPC fate, resulting in cell death and senescence in the dorsal and ventral subsets, respectively. Such alternative fates are associated with distinct developmental origins of OPCs, and with a different activation of NRF2-mediated anti-oxidant responses. These data indicate that, upon injury, dorsal and ventral OPC subsets show functional and molecular diversity that can make them differentially vulnerable to pathological conditions associated with DNA damage., (© 2022. The Author(s).)

Medulloblastoma (MB) and gliomas are the most frequent high-grade brain tumors (HGBT) in children and adulthood, respectively. The general treatment for these tumors consists in surgery, followed by radiotherapy and chemotherapy. Despite the improvement in patient survival, these therapies are only partially effective, and many patients still die. In the last decades, microtubules have emerged as interesting molecular targets for HGBT, as various microtubule targeting agents (MTAs) have been developed and tested pre-clinically and clinically with encouraging results. Nevertheless, these treatments produce relevant side effects since they target microtubules in normal as well as in cancerous cells. A possible strategy to overcome this toxicity could be to target proteins that control microtubule dynamics but are required by HGBT cells much more than in normal cell types. The genes mutated in primary hereditary microcephaly (MCPH) are ubiquitously expressed in proliferating cells, but under normal conditions are selectively required during brain development, in neural progenitors. There is evidence that MB and glioma cells share molecular profiles with progenitors of cerebellar granules and of cortical radial glia cells, in which MCPH gene functions are fundamental. Moreover, several studies indicate that MCPH genes are required for HGBT expansion. Among the 25 known MCPH genes, we focus this review on KNL1, ASPM, CENPE, CITK and KIF14, which have been found to control microtubule stability during cell division. We summarize the current knowledge about the molecular basis of their interaction with microtubules. Moreover, we will discuss data that suggest these genes are promising candidates as HGBT-specific targets., (© 2021. The Author(s).)

Goldberg-Shprintzen disease (GOSHS) is a rare microcephaly syndrome accompanied by intellectual disability, dysmorphic facial features, peripheral neuropathy and Hirschsprung disease. It is associated with recessive mutations in the gene encoding kinesin family member 1-binding protein (KIF1BP, also known as KIFBP). The encoded protein regulates axon microtubules dynamics, kinesin attachment and mitochondrial biogenesis, but it is not clear how its loss could lead to microcephaly. We identified KIF1BP in the interactome of citron kinase (CITK, also known as CIT), a protein produced by the primary hereditary microcephaly 17 (MCPH17) gene. KIF1BP and CITK interact under physiological conditions in mitotic cells. Similar to CITK, KIF1BP is enriched at the midbody ring and is required for cytokinesis. The association between KIF1BP and CITK can be influenced by CITK activity, and the two proteins may antagonize each other for their midbody localization. KIF1BP knockdown decreases microtubule stability, increases KIF23 midbody levels and impairs midbody localization of KIF14, as well as of chromosome passenger complex. These data indicate that KIF1BP is a CITK interactor involved in midbody maturation and abscission, and suggest that cytokinesis failure may contribute to the microcephaly phenotype observed in GOSHS., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Spinal cord injury (SCI) affects 6 million people worldwide with no available treatment. Despite research advances, the inherent poor regeneration potential of the central nervous system remains a major hurdle. Small RNAs (sRNAs) 19-33 nucleotides in length are a set of non-coding RNA molecules that regulate gene expression and have emerged as key players in regulating cellular events occurring after SCI. Here we profiled a class of sRNA known as microRNAs (miRNAs) following SCI in the cortex where the cell bodies of corticospinal motor neurons are located. We identified miR-7b-3p as a candidate target given its significant upregulation after SCI in vivo and we screened by miRWalk PTM the genes predicted to be targets of miR-7b-3p (among which we identified Wipf2 , a gene regulating neurite extension). Moreover, 16 genes, involved in neural regeneration and potential miR-7b-3p targets, were found to be downregulated in the cortex following SCI. We also analysed miR-7b-3p function during cortical neuron development in vitro : we observed that the overexpression of miR-7b-3p was important (1) to maintain neurons in a more immature and, likely, plastic neuronal developmental phase and (2) to contrast the apoptotic pathway; however, in normal conditions it did not affect the Wipf2 expression. On the contrary, the overexpression of miR-7b-3p upon in vitro oxidative stress condition (mimicking the SCI environment) significantly reduced the expression level of Wipf2, as observed in vivo , confirming it as a direct miR-7b-3p target. Overall, these data suggest a dual role of miR-7b-3p: (i) the induction of a more plastic neuronal condition/phase, possibly at the expense of the axon growth, (ii) the neuroprotective role exerted through the inhibition of the apoptotic cascade. Increasing the miR-7b-3p levels in case of SCI could reactivate in adult neurons silenced developmental programmes, supporting at the same time the survival of the axotomised neurons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghibaudi, Boido, Green, Signorino, Berto, Pourshayesteh, Singh, Di Cunto, Dalmay and Vercelli.)

Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE's inhibition could be a therapeutic strategy for MB treatment.

Mutations in MECP2 cause several neurological disorders of which Rett syndrome (RTT) represents the best-defined condition. Although mainly working as a transcriptional repressor, MeCP2 is a multifunctional protein revealing several activities, the involvement of which in RTT remains obscure. Besides being mainly localized in the nucleus, MeCP2 associates with the centrosome, an organelle from which primary cilia originate. Primary cilia function as "sensory antennae" protruding from most cells, and a link between primary cilia and mental illness has recently been reported. We herein demonstrate that MeCP2 deficiency affects ciliogenesis in cultured cells, including neurons and RTT fibroblasts, and in the mouse brain. Consequently, the cilium-related Sonic Hedgehog pathway, which is essential for brain development and functioning, is impaired. Microtubule instability participates in these phenotypes that can be rescued by HDAC6 inhibition together with the recovery of RTT-related neuronal defects. Our data indicate defects of primary cilium as a novel pathogenic mechanism that by contributing to the clinical features of RTT might impact on proper cerebellum/brain development and functioning, thus providing a novel therapeutic target., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The Citron protein was originally identified for its capability to specifically bind the active form of RhoA small GTPase, leading to the simplistic hypothesis that it may work as a RhoA downstream effector in actin remodeling. More than two decades later, a much more complex picture has emerged. In particular, it has become clear that in animals, and especially in mammals, the functions of the Citron gene ( CIT ) are intimately linked to many aspects of central nervous system (CNS) development and function, although the gene is broadly expressed. More specifically, CIT encodes two main isoforms, Citron-kinase (CIT-K) and Citron-N (CIT-N), characterized by complementary expression pattern and different functions. Moreover, in many of their activities, CIT proteins act more as upstream regulators than as downstream effectors of RhoA. Finally it has been found that, besides working through actin, CIT proteins have many crucial functional interactions with the microtubule cytoskeleton and may directly affect genome stability. In this review, we will summarize these advances and illustrate their actual or potential relevance for CNS diseases, including microcephaly and psychiatric disorders.

Medulloblastoma (MB) is the most common malignant brain tumor in children, and it is classified into four biological subgroups: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. The current treatment is surgery, followed by irradiation and chemotherapy. Unfortunately, these therapies are only partially effective. Citron kinase protein (CITK) has been proposed as a promising target for SHH MB, whose inactivation leads to DNA damage and apoptosis. D283 and D341 cell lines (Group 3/Group 4 MB) were silenced with established siRNA sequences against CITK, to assess the direct effects of its loss. Next, D283, D341, ONS-76 and DAOY cells were treated with ionizing radiation (IR) or cisplatin in combination with CITK knockdown. CITK depletion impaired proliferation and induced cytokinesis failure and apoptosis of G3/G4 MB cell lines. Furthermore, CITK knockdown produced an accumulation of DNA damage, with reduced RAD51 nuclear levels. Association of IR or cisplatin with CITK depletion strongly impaired the growth potential of all tested MB cells. These results indicate that CITK inactivation could prevent the expansion of G3/G4 MB and increase their sensitivity to DNA-damaging agents, by impairing homologous recombination. We suggest that CITK inhibition could be broadly associated with IR and adjuvant therapy in MB treatment., Competing Interests: The authors declare no conflicts of interest.

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

The inhibitory effect of large tumor suppressor kinase (LATS1/2) on the activity of the oncoprotein yes-associated protein (YAP) is crucial to maintain tissue homeostasis. Proteomic studies have identified several new regulators of this process. Recently, citron kinase (CIT) was listed as a potential binding candidate of Hippo-related components, suggesting a new connection between CIT and the Hippo pathway. Aside from CIT's role in cytokinesis, the molecular crosstalk between CIT and the Hippo pathway is largely unknown. Here, we demonstrate a role for CIT as a scaffold protein linking LATS2 and YAP. More importantly, CIT interacts with LATS2 to directly suppress LATS2 phosphorylation at the hydrophobic motif-targeted by MST1, leading to LATS2 inactivation and YAP activation. By studying their genetic interactions, we found that Sticky, the CIT homolog in Drosophila melanogaster, functions with Warts to control Drosophila eye development. Together, our study confirms citron kinase as a novel regulator of the Hippo pathway., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Down Syndrome (DS) is the most common genetic disorder associated with intellectual disability (ID). Excitatory neurons of DS patients and mouse models show decreased size of dendritic field and reduction of spine density. Whether these defects are caused by cell autonomous alterations or by abnormal multicellular circuitry is still unknown. In this work, we explored this issue by culturing cortical neurons obtained from two mouse models of DS: the widely used Ts65Dn and the less characterized Ts2Cje. We observed that, in the in vitro conditions, axon specification and elongation, as well as dendritogenesis, take place without evident abnormalities, indicating that the initial phases of neuronal differentiation do not suffer from the presence of an imbalanced genetic dosage. Conversely, our analysis highlighted differences between trisomic and euploid neurons in terms of reduction of spine density, in accordance with in vivo data obtained by other groups, proposing the presence of a cell-intrinsic malfunction. This work suggests that the characteristic morphological defects of DS neurons are likely to be caused by the possible combination of cell-intrinsic defects together with cell-extrinsic cues. Additionally, our data support the possibility of using the more sustainable line Ts2Cje as a standard model for the study of DS., (Copyright © 2019 Chiotto, Migliorero, Pallavicini, Bianchi, Gai, Di Cunto and Berto.)

Glioblastoma multiforme and medulloblastoma are the most frequent high-grade brain tumors in adults and children, respectively. Standard therapies for these cancers are mainly based on surgical resection, radiotherapy, and chemotherapy. However, intrinsic or acquired resistance to treatment occurs almost invariably in the first case, and side effects are unacceptable in the second. Therefore, the development of new, effective drugs is a very important unmet medical need. A critical requirement for developing such agents is to identify druggable targets required for the proliferation or survival of tumor cells, but not of other cell types. Under this perspective, genes mutated in congenital microcephaly represent interesting candidates. Congenital microcephaly comprises a heterogeneous group of disorders in which brain volume is reduced, in the absence or presence of variable syndromic features. Genetic studies have clarified that most microcephaly genes encode ubiquitous proteins involved in mitosis and in maintenance of genomic stability, but the effects of their inactivation are particularly strong in neural progenitors. It is therefore conceivable that the inhibition of the function of these genes may specifically affect the proliferation and survival of brain tumor cells. Microcephaly genes encode for a few kinases, including CITK, PLK4, AKT3, DYRK1A, and TRIO. In this review, we summarize the evidence indicating that the inhibition of these molecules could exert beneficial effects on different aspects of brain cancer treatment., Competing Interests: The authors declare no conflict of interest.

We report an apparently sporadic amyotrophic lateral sclerosis patient carrying a heterozygous novel frameshift SOD1 mutation (p.Ser108LeufsTer15), predicted to cause a premature protein truncation. RT-PCR analysis of SOD1 mRNA and SDS-PAGE/Western blot analysis of PBMC demonstrated that mRNA from the mutant allele is expressed at levels similar to those of the wild-type allele, but the truncated protein is undetectable also in the insoluble fraction and after proteasome inhibition. Accordingly, the dismutation activity in erythrocytes is halved. Thus, the pathogenic mechanism associated with this mutation might be based on an insufficient activity of SOD1 that would make motor neurons more vulnerable to oxidative injury. However, it cannot be excluded that p.Ser108LeufsTer15 SOD1 is present in the nervous tissue and, being less charged and hence having less repulsive forces than the wild-type protein, may trigger toxic mechanisms as a consequence of its propensity to aggregate., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Epilepsy is a complex clinical condition characterized by repeated spontaneous seizures. Seizures have been linked to multiple drivers including DNA damage accumulation. Investigation of epilepsy physiopathology in humans imposes ethical and practical limitations, for this reason model systems are mostly preferred. Among animal models, mouse mutants are particularly valuable since they allow conjoint behavioral, organismal, and genetic analyses. Along with this, since aging has been associated with higher frequency of seizures, prematurely aging mice, simulating human progeroid diseases, offer a further useful modeling element as they recapitulate aging over a short time-window. Here we report on a mouse mutant with progeroid traits that displays repeated spontaneous seizures. Mutant mice were produced by reducing the expression of the gene Ft1 ( AKTIP in humans). In vitro , AKTIP/Ft1 depletion causes telomere aberrations, DNA damage, and cell senescence. AKTIP/Ft1 interacts with lamins, which control nuclear architecture and DNA function. Premature aging defects of Ft1 mutant mice include skeletal alterations and lipodystrophy. The epileptic behavior of Ft1 mutant animals was age and sex linked. Seizures were observed in 18 mutant mice (23.6% of aged ≥ 21 weeks), at an average frequency of 2.33 events/mouse. Time distribution of seizures indicated non-random enrichment of seizures over the follow-up period, with 75% of seizures happening in consecutive weeks. The analysis of epileptic brains did not reveal overt brain morphological alterations or severe neurodegeneration, however, Ft1 reduction induced expression of the inflammatory markers IL-6 and TGF-β. Importantly, Ft1 mutant mice with concomitant genetic reduction of the guardian of the genome, p53, showed no seizures or inflammatory marker activation, implicating the DNA damage response into these phenotypes. This work adds insights into the connection among DNA damage, brain function, and aging. In addition, it further underscores the importance of model organisms for studying specific phenotypes, along with permitting the analysis of genetic interactions at the organismal level.

Maintenance of genome stability is a crucial cellular function for normal mammalian development and physiology. However, despite the general relevance of this process, genome stability alteration due to genetic or non-genetic conditions has a particularly profound impact on the developing cerebral cortex. In this review, we will analyze the main pathways involved in maintenance of genome stability, the consequences of their alterations with regard to central nervous system development, as well as the possible molecular and cellular basis of this specificity.

Abscission is the final step of cytokinesis whereby the intercellular bridge (ICB) linking the two daughter cells is cut. The ICB contains a structure called the midbody, required for the recruitment and organization of the abscission machinery. Final midbody severing is mediated by formation of secondary midbody ingression sites, where the ESCRT III component CHMP4B is recruited to mediate membrane fusion. It is presently unknown how cytoskeletal elements cooperate with CHMP4B to mediate abscission. Here, we show that F-actin is associated with midbody secondary sites and is necessary for abscission. F-actin localization at secondary sites depends on the activity of RhoA and on the abscission regulator citron kinase (CITK). CITK depletion accelerates loss of F-actin proteins at the midbody and subsequent cytokinesis defects are reversed by restoring actin polymerization. Conversely, midbody hyperstabilization produced by overexpression of CITK and ANLN is reversed by actin depolymerization. CITK is required for localization of F-actin and ANLN at the abscission sites, as well as for CHMP4B recruitment. These results indicate that control of actin dynamics downstream of CITK prepares the abscission site for the final cut., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Systemic anaplastic large cell lymphomas (ALCL) are a category of T-cell non-Hodgkin's lymphomas which can be divided into anaplastic lymphoma kinase (ALK) positive and ALK negative subgroups, based on ALK gene rearrangements. Among several pathways aberrantly activated in ALCL, the constitutive activation of signal transducer and activator of transcription 3 (STAT3) is shared by all ALK positive ALCL and has been detected in a subgroup of ALK negative ALCL. To discover essential mediators of STAT3 oncogenic activity that may represent feasible targets for ALCL therapies, we combined gene expression profiling analysis and RNA interference functional approaches. A shRNA screening of STAT3-modulated genes identified interferon regulatory factor 4 (IRF4) as a key driver of ALCL cell survival. Accordingly, ectopic IRF4 expression partially rescued STAT3 knock-down effects. Treatment with immunomodulatory drugs (IMiDs) induced IRF4 down regulation and resulted in cell death, a phenotype rescued by IRF4 overexpression. However, the majority of ALCL cell lines were poorly responsive to IMiDs treatment. Combination with JQ1, a bromodomain and extra-terminal (BET) family antagonist known to inhibit MYC and IRF4, increased sensitivity to IMiDs. Overall, these results show that IRF4 is involved in STAT3-oncogenic signaling and its inhibition provides alternative avenues for the design of novel/combination therapies of ALCL., Competing Interests: The authors have no conflicts of interest.

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins., (© 2016 The Authors.)

Primary microcephaly is a neurodevelopmental disorder that is caused by a reduction in brain size as a result of defects in the proliferation of neural progenitor cells during development. Mutations in genes encoding proteins that localize to the mitotic spindle and centrosomes have been implicated in the pathogenicity of primary microcephaly. In contrast, the contractile ring and midbody required for cytokinesis, the final stage of mitosis, have not previously been implicated by human genetics in the molecular mechanisms of this phenotype. Citron kinase (CIT) is a multi-domain protein that localizes to the cleavage furrow and midbody of mitotic cells, where it is required for the completion of cytokinesis. Rodent models of Cit deficiency highlighted the role of this gene in neurogenesis and microcephaly over a decade ago. Here, we identify recessively inherited pathogenic variants in CIT as the genetic basis of severe microcephaly and neonatal death. We present postmortem data showing that CIT is critical to building a normally sized human brain. Consistent with cytokinesis defects attributed to CIT, multinucleated neurons were observed throughout the cerebral cortex and cerebellum of an affected proband, expanding our understanding of mechanisms attributed to primary microcephaly., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mutations in MECP2 cause a broad spectrum of neuropsychiatric disorders of which Rett syndrome represents the best defined condition. Both neuronal and non-neuronal functions of the methyl-binding protein underlie the related pathologies. Nowadays MeCP2 is recognized as a multifunctional protein that modulates its activity depending on its protein partners and posttranslational modifications. However, we are still missing a comprehensive understanding of all MeCP2 functions and their involvement in the related pathologies. The study of human mutations often offers the possibility of clarifying the functions of a protein. Therefore, we decided to characterize a novel MeCP2 phospho-isoform (Tyr-120) whose relevance was suggested by a Rett syndrome patient carrying a Y120D substitution possibly mimicking a constitutively phosphorylated state. Unexpectedly, we found MeCP2 and its Tyr-120 phospho-isoform enriched at the centrosome both in dividing and postmitotic cells. The molecular and functional connection of MeCP2 to the centrosome was further reinforced through cellular and biochemical approaches. We show that, similar to many centrosomal proteins, MeCP2 deficiency causes aberrant spindle geometry, prolonged mitosis, and defects in microtubule nucleation. Collectively, our data indicate a novel function of MeCP2 that might reconcile previous data regarding the role of MeCP2 in cell growth and cytoskeleton stability and that might be relevant to understand some aspects of MeCP2-related conditions. Furthermore, they link the Tyr-120 residue and its phosphorylation to cell division, prompting future studies on the relevance of Tyr-120 for cortical development., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)