Gcap14 is a microtubule plus-end-tracking protein coordinating microtubule-actin crosstalk during neurodevelopment.

Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.

AMP-activated protein kinase regulates cytoplasmic dynein behavior and contributes to neuronal migration in the developing neocortex.

The microtubule motor cytoplasmic dynein contributes to radial migration of newborn pyramidal neurons in the developing neocortex. Here, we show that AMP-activated protein kinase (AMPK) mediates the nucleus-centrosome coupling, a key process for radial neuronal migration that relies on dynein. Depletion of the catalytic subunit of AMPK in migrating neurons impairs this coupling as well as neuronal migration. AMPK shows overlapping subcellular distribution with cytoplasmic dynein and the two proteins interact with each other. Pharmacological inhibition or activation of AMPK modifies the phosphorylation states of dynein intermediate chain (DIC) and dynein functions. Furthermore, AMPK phosphorylates DIC at Ser81. Expression of a phospho-resistant mutant of DIC retards neuronal migration in a similar way to AMPK depletion. Conversely, expression of the phospho-mimetic mutant of DIC alleviates impaired neuronal migration caused by AMPK depletion. Thus, AMPK-regulated dynein function via Ser81 DIC phosphorylation is crucial for radial neuronal migration.

The LPA-LPA4 axis is required for establishment of bipolar morphology and radial migration of newborn cortical neurons.

Newborn neurons in the developing neocortex undergo radial migration, a process that is coupled with their precise passage from multipolar to bipolar shape. The cell-extrinsic signals that govern this transition are, however, poorly understood. Here, we find that lysophosphatidic acid (LPA) signaling contributes to the establishment of a bipolar shape in mouse migratory neurons through LPA receptor 4 (LPA4). LPA4 is robustly expressed in migratory neurons. LPA4-depleted neurons show impaired multipolar-to-bipolar transition and become arrested in their migration. Further, LPA4-mediated LPA signaling promotes formation of the pia-directed process in primary neurons overlaid on neocortical slices. In addition, LPA4 depletion is coupled with altered actin organization as well as with destabilization of the F-actin-binding protein filamin A (FlnA). Finally, overexpression of FlnA rescues the morphology and migration defects of LPA4-depleted neurons. Thus, the LPA-LPA4 axis regulates bipolar morphogenesis and radial migration of newborn cortical neurons via remodeling of the actin cytoskeleton.

Reelin Improves Cognition and Extends the Lifespan of Mutant Ndel1 Mice with Postnatal CA1 Hippocampus Deterioration.

The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10 weeks). Here we report that Ndel1 CKO mice (of both sexes) experience spatial learning and memory deficits that are associated with deregulation of neuronal cell adhesion, plasticity and neurotransmission genes, as assessed by genome-wide transcriptome analysis of the hippocampus. Importantly, a single injection of Reelin protein in the hippocampus of Ndel1 CKO mice improves spatial learning and memory function and this is correlated with reduced intrinsic hyperexcitability of CA1 pyramidal neurons, and normalized gene deregulation in the hippocampus. Strikingly, when treated with Reelin, Ndel1 CKO animals that die from an epileptic phenotype, live twice as long as nontreated, or vehicle-treated CKO animals. Thus, Reelin confers striking beneficial effects in the CA1 hippocampus, and at both behavioral and organismal levels.

Ndel1 and Reelin Maintain Postnatal CA1 Hippocampus Integrity.

UNLABELLED: How the integrity of laminar structures in the postnatal brain is maintained impacts neuronal functions. Ndel1, the mammalian homolog of NuDE from the filamentous fungus Aspergillus nidulans, is an atypical microtubule (MT)-associated protein that was initially investigated in the contexts of neurogenesis and neuronal migration. Constitutive knock-out mice for Ndel1 are embryonic lethal, thereby necessitating the creation a conditional knock-out to probe the roles of Ndel1 in postnatal brains. Here we report that CA1 pyramidal neurons from mice postnatally lacking Ndel1 (Ndel1 conditional knock-out) exhibit fragmented MTs, dendritic/synaptic pathologies, are intrinsically hyperexcitable and undergo dispersion independently of neuronal migration defect. Secondary to the pyramidal cell changes is the decreased inhibitory drive onto pyramidal cells from interneurons. Levels of the glycoprotein Reelin that regulates MTs, neuronal plasticity, and cell compaction are significantly reduced in hippocampus of mutant mice. Strikingly, a single injection of Reelin into the hippocampus of Ndel1 conditional knock-out mice ameliorates ultrastructural, cellular, morphological, and anatomical CA1 defects. Thus, Ndel1 and Reelin contribute to maintain postnatal CA1 integrity. SIGNIFICANCE STATEMENT: The significance of this study rests in the elucidation of a role for Nde1l and Reelin in postnatal CA1 integrity using a new conditional knock-out mouse model for the cytoskeletal protein Ndel1, one that circumvents the defects associated with neuronal migration and embryonic lethality. Our study serves as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function, and the significance of decreased levels of Ndel1 and Reelin observed in patients with neurological disorders.

DYRK1A overexpression enhances STAT activity and astrogliogenesis in a Down syndrome mouse model.

Down syndrome (DS) arises from triplication of genes on human chromosome 21 and is associated with anomalies in brain development such as reduced production of neurons and increased generation of astrocytes. Here, we show that differentiation of cortical progenitor cells into astrocytes is promoted by DYRK1A, a Ser/Thr kinase encoded on human chromosome 21. In the Ts1Cje mouse model of DS, increased dosage of DYRK1A augments the propensity of progenitors to differentiate into astrocytes. This tendency is associated with enhanced astrogliogenesis in the developing neocortex. We also find that overexpression of DYRK1A upregulates the activity of the astrogliogenic transcription factor STAT in wild-type progenitors. Ts1Cje progenitors exhibit elevated STAT activity, and depletion of DYRK1A in these cells reverses the deregulation of STAT. In sum, our findings indicate that potentiation of the DYRK1A-STAT pathway in progenitors contributes to aberrant astrogliogenesis in DS.

Somato-dendritic decoupling as a novel mechanism for protracted cortical maturation.

BACKGROUND: Both human and animal data indicate that disruption of the endogenously slow maturation of temporal association cortical (TeA) networks is associated with abnormal higher order cognitive development. However, the neuronal mechanisms underlying the endogenous maturation delay of the TeA are poorly understood. RESULTS: Here we report a novel form of developmental plasticity that is present in the TeA. It was found that deep layer TeA neurons, but not hippocampal or primary visual neurons, exist in a protracted 'embryonic-like' state through a mechanism involving reduced somato-dendritic communication and a non-excitable somatic membrane. This mechanism of neural inactivity is present in intact tissue and shows a remarkable transition into an active somato-dendritically coupled state. The quantity of decoupled cells diminishes in a protracted and age-dependent manner, continuing into adolescence. CONCLUSIONS: Based on our data, we propose a model of neural plasticity through which protracted compartmentalization and decoupling in somato-dendritic signalling plays a key role in controlling how excitable neurons are incorporated into recurrent cortical networks independent of neurogenesis.

Zac1 Regulates the Differentiation and Migration of Neocortical Neurons via Pac1.

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. Significance statement: Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader-Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.

Phosphorylation of targeting protein for Xenopus kinesin-like protein 2 (TPX2) at threonine 72 in spindle assembly.

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.

Disrupted-in-schizophrenia 1 (DISC1) regulates dysbindin function by enhancing its stability.

Dysbindin and DISC1 are schizophrenia susceptibility factors playing roles in neuronal development. Here we show that the physical interaction between dysbindin and DISC1 is critical for the stability of dysbindin and for the process of neurite outgrowth. We found that DISC1 forms a complex with dysbindin and increases its stability in association with a reduction in ubiquitylation. Furthermore, knockdown of DISC1 or expression of a deletion mutant, DISC1 lacking amino acid residues 403-504 of DISC1 (DISC1(Δ403-504)), effectively decreased levels of endogenous dysbindin. Finally, the neurite outgrowth defect induced by knockdown of DISC1 was partially reversed by coexpression of dysbindin. Taken together, these results indicate that dysbindin and DISC1 form a physiologically functional complex that is essential for normal neurite outgrowth.

The G protein-coupled receptor GPRC5B contributes to neurogenesis in the developing mouse neocortex.

Neural progenitor cells in the developing brain give rise to neurons and glia. Multiple extrinsic signalling molecules and their cognate membrane receptors have been identified to control neural progenitor fate. However, a role for G protein-coupled receptors in cell fate decisions in the brain remains largely putative. Here we show that GPRC5B, which encodes an orphan G protein-coupled receptor, is present in the ventricular surface of cortical progenitors in the mouse developing neocortex and is required for their neuronal differentiation. GPRC5B-depleted progenitors fail to adopt a neuronal fate and ultimately become astrocytes. Furthermore, GPRC5B-mediated signalling is associated with the proper regulation of ß-catenin signalling, a pathway crucial for progenitor fate decision. Our study uncovers G protein-coupled receptor signalling in the neuronal fate determination of cortical progenitors.

p600/UBR4 in the central nervous system.

A decade ago, the large 600 kDa mammalian protein p600 (also known as UBR4) was discovered as a multifunctional protein with roles in anoikis, viral transformation and protein degradation. Recently, p600 has emerged as a critical protein in the mammalian brain with roles in neurogenesis, neuronal migration, neuronal signaling and survival. How p600 integrates these apparently unrelated functions to maintain tissue homeostasis and murine survival remains unclear. The common molecular basis underlying many of the actions of p600 suggests, however, certain conservation and transposition of these functions across systems. In this review, we summarize the central nervous system functions of p600 and propose new perspectives on its biological complexity in neuronal physiology and neurological diseases.

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy.

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.

Neuroprotective role of the basic leucine zipper transcription factor NFIL3 in models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Here we show that the basic leucine zipper transcription factor NFIL3 (also called E4BP4) confers neuroprotection in models of ALS. NFIL3 is up-regulated in primary neurons challenged with neurotoxic insults and in a mouse model of ALS. Overexpression of NFIL3 attenuates excitotoxic neuronal damage and protects neurons against neurodegeneration in a cell-based ALS model. Conversely, reduction of NFIL3 exacerbates neuronal demise in adverse conditions. Transgenic neuronal expression of NFIL3 in ALS mice delays disease onset and attenuates motor axon and neuron degeneration. These results suggest that NFIL3 plays a neuroprotective role in neurons and constitutes a potential therapeutic target for neurodegeneration.

TPX2: of spindle assembly, DNA damage response, and cancer.

For more than 15 years, TPX2 has been studied as a factor critical for mitosis and spindle assembly. These functions of TPX2 are attributed to its Ran-regulated microtubule-associated protein properties and to its control of the Aurora A kinase. Overexpressed in cancers, TPX2 is being established as marker for the diagnosis and prognosis of malignancies. During interphase, TPX2 resides preferentially in the nucleus where its function had remained elusive until recently. The latest finding that TPX2 plays a role in amplification of the DNA damage response, combined with the characterization of TPX2 knockout mice, open new perspectives to understand the biology of this protein. This review provides an historic overview of the discovery of TPX2 and summarizes its cytoskeletal and signaling roles with relevance to cancer therapies. Finally, the review aims to reconcile discrepancies between the experimental and pathological effects of TPX2 overexpression and advances new roles for compartmentalized TPX2.

A Ca2+-dependent mechanism of neuronal survival mediated by the microtubule-associated protein p600.

In acute and chronic neurodegeneration, Ca(2+) mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca(2+) influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca(2+) release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca(2+)-induced death pathways through regulation of Ca(2+) homeostasis and signaling.

p600 stabilizes microtubules to prevent the aggregation of CaMKIIα during photoconductive stimulation.

The large microtubule-associated/Ca(2+)-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca(2+) dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca(2+).

Brain-body mechanisms contribute to sexual dimorphism in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common form of human motor neuron disease. It is characterized by the progressive degeneration of upper and lower motor neurons, leading to generalized motor weakness and, ultimately, respiratory paralysis and death within 3-5 years. The disease is shaped by genetics, age, sex and environmental stressors, but no cure or routine biomarkers exist for the disease. Male individuals have a higher propensity to develop ALS, and a different manifestation of the disease phenotype, than female individuals. However, the mechanisms underlying these sex differences remain a mystery. In this Review, we summarize the epidemiology of ALS, examine the sexually dimorphic presentation of the disease and highlight the genetic variants and molecular pathways that might contribute to sex differences in humans and animal models of ALS. We advance the idea that sexual dimorphism in ALS arises from the interactions between the CNS and peripheral organs, involving vascular, metabolic, endocrine, musculoskeletal and immune systems, which are strikingly different between male and female individuals. Finally, we review the response to treatments in ALS and discuss the potential to implement future personalized therapeutic strategies for the disease.

Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates γ-histone 2AX (γ-H2AX) levels upon ionizing radiation.

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin.

Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca(2+) dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin.