Cortical Layer Inversion and Deregulation of Reelin Signaling in the Absence of SOCS6 and SOCS7.

Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.

Engineered Exosomes as Vehicles for Biologically Active Proteins.

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination, indicating functional delivery of the protein to recipient cells. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases.

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells.

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.

Rab5 and Ndfip1 are involved in Pten ubiquitination and nuclear trafficking.

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC-CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri-nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.

Nedd4 family interacting protein 1 (Ndfip1) is required for ubiquitination and nuclear trafficking of BRCA1-associated ATM activator 1 (BRAT1) during the DNA damage response.

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1.

Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates.

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved.

PTEN secretion in exosomes.

PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis.

The zinc finger transcription factor RP58 negatively regulates Rnd2 for the control of neuronal migration during cerebral cortical development.

The zinc finger transcription factor RP58 (also known as ZNF238) regulates neurogenesis of the mouse neocortex and cerebellum (Okado et al. 2009; Xiang et al. 2011; Baubet et al. 2012; Ohtaka-Maruyama et al. 2013), but its mechanism of action remains unclear. In this study, we report a cell-autonomous function for RP58 during the differentiation of embryonic cortical projection neurons via its activities as a transcriptional repressor. Disruption of RP58 expression alters the differentiation of immature neurons and impairs their migration and positioning within the mouse cerebral cortex. Loss of RP58 within the embryonic cortex also leads to elevated mRNA for Rnd2, a member of the Rnd family of atypical RhoA-like GTPase proteins important for cortical neuron migration (Heng et al. 2008). Mechanistically, RP58 represses transcription of Rnd2 via binding to a 3'-regulatory enhancer in a sequence-specific fashion. Using reporter assays, we found that RP58 repression of Rnd2 is competed by proneural basic helix-loop-helix transcriptional activators. Finally, our rescue experiments revealed that negative regulation of Rnd2 by RP58 was important for cortical cell migration in vivo. Taken together, these studies demonstrate that RP58 is a key player in the transcriptional control of cell migration in the developing cerebral cortex.

Exogenous Reelin modifies the migratory behavior of neurons depending on cortical location.

Malformations of cortical development can arise when projection neurons generated in the germinal zones fail to migrate properly into the cortical plate. This process is critically dependent on the Reelin glycoprotein, which when absent leads to an inversion of cortical layers and blurring of borders. Reelin has other functions including supporting neuron migration and maintaining their trajectories; however, the precise role on glial fiber-dependent or -independent migration of neurons remains controversial. In this study, we wish to test the hypothesis that migrating cortical neurons at different levels of the cortical wall have differential responses to Reelin. We exposed neurons migrating across the cortical wall to exogenous Reelin and monitored their migratory behavior using time-lapse imaging. Our results show that, in the germinal zones, exogenous Reelin retarded neuron migration and altered their trajectories. This behavior is in contrast to the response of neurons located in the intermediate zone (IZ), possibly because Reelin receptors are not expressed in this zone. In the reeler cortex, Reelin receptors are expressed in the IZ and exposure to exogenous Reelin was able to rescue the migratory defect. These studies demonstrate that migrating neurons have nonequivalent responses to Reelin depending on their location within the cortical wall.

Ndfip1 is required for the development of pyramidal neuron dendrites and spines in the neocortex.

Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.

Perturbations in cortical development and neuronal network excitability arising from prenatal exposure to benzodiazepines in mice.

During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.

Cellular up-regulation of Nedd4 family interacting protein 1 (Ndfip1) using low levels of bioactive cobalt complexes.

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.

Altered speeds and trajectories of neurons migrating in the ventricular and subventricular zones of the reeler neocortex.

The Reelin signaling pathway is essential for proper cortical development, but it is unclear to whether Reelin function is primarily important for cortical layering or neuron migration. It has been proposed that Reelin is perhaps required only for somal translocation but not glial-dependent locomotion. This implies that the location of neurons responding to Reelin is restricted to the outer regions of the cortical plate (CP). To determine whether Reelin is required for migration outside of the CP, we used time-lapse imaging to track the behavior of cells undergoing locomotion in the germinal zones. We focused on the migratory activity in the ventricular/subventricular zones where the first transition of bipolar to multipolar migration occurs and where functional Reelin receptors are known to be expressed. Despite Reelin loss, neurons had no difficulty in undergoing radial migration and indeed displayed greater migratory speed. Additionally, compared with the wild-type, reeler neurons displayed altered trajectories with greater deviation from a radial path. These results suggest that Reelin loss has early consequences for migration in the germinal zones that are portrayed as defective radial trajectories and migratory speeds. Together, these abnormalities can give rise to the increased cell dispersion observed in the reeler cortex.

Spatiotemporal regulation of multiple overlapping sense and novel natural antisense transcripts at the Nrgn and Camk2n1 gene loci during mouse cerebral corticogenesis.

Nrgn and Camk2n1 are highly expressed in the brain and play an important role in synaptic long-term potentiation via regulation of Ca(2+)/calmodulin-dependent protein kinase II. We have shown that the gene loci for these 2 proteins are actively transcribed in the adult cerebral cortex and feature multiple overlapping transcripts in both the sense and antisense orientations with alternative polyadenylation. These transcripts were upregulated in the adult compared with embryonic and P1.5 mouse cerebral cortices, and transcripts with different 3' untranslated region lengths showed differing expression profiles. In situ hybridization (ISH) analysis revealed spatiotemporal regulation of the Nrgn and Camk2n1 sense and natural antisense transcripts (NATs) throughout cerebral corticogenesis. In addition, we also demonstrated that the expression of these transcripts was organ-specific. Both Nrgn and Camk2n1 sense and NATs were also upregulated in differentiating P19 teratocarcinoma cells. RNA fluorescent ISH analysis confirmed the capability of these NATs to form double-stranded RNA aggregates with the sense transcripts in the cytoplasm of cells obtained from the brain. We propose that the differential regulation of multiple sense and novel overlapping NATs at the Nrgn and Camk2n1 loci will increase the diversity of posttranscriptional regulation, resulting in cell- and time-specific regulation of their gene products during cerebral corticogenesis and function.

Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons.

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4-2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1(-/-) with Ndfip1(+/+) mice exposed to Fe reveals that Ndfip1(-/-) brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.

Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons.

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.

Deletion of CREB1 from the dorsal telencephalon reduces motivational properties of cocaine.

Transcriptional changes in neurons underpin the long-lived cellular response to environmental stimuli, and cAMP-responsive element-binding protein (CREB1) has been implicated in this process. Exposure to psychostimulants such as cocaine results in persistent neuronal plasticity in cortical circuitry that likely modulates the motivation to use the drug again. To examine whether CREB1 in cortical glutamatergic neurons was implicated in cocaine use, we developed conditional CREB1 mutants that exhibit ablation of functional CREB1 in the cortex and hippocampus. Here we report that CREB1 mutants show normal locomotor responses to acute and chronic cocaine and develop a place preference for cocaine. However, CREB1 mutants demonstrate a diminished drive to self-administer cocaine under operant conditions. We conclude that there is a specific role for CREB1 in telencephalic glutamatergic neurons regulating the motivational properties of cocaine.

Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras.

Reelin is an important protein that is indispensable for cortical lamination. In the absence of Reelin, cortical layers fail to form due to inappropriate neuron migration and positioning. The inversion of cortical layers is attributed to failure of neurons to migrate past earlier-generated neurons although how Reelin-insufficiency causes this is unclear. The issue is complicated by recent studies showing that very little Reelin is required for cortical layering. To test how variation in the number of Reelin-producing cells is linked to cortical lamination, we have employed Reelin(+/+) <--> Reelin(-/-) chimeras in which the number of Reelin-expressing neurons is adjusted. We found that the Reeler phenotype was rescued in chimeras with a large contribution of Reelin(+/+) neurons; conversely in chimeras with a weak contribution by Reelin(+/+) neurons, the mutant phenotype remained. However, increasing the number of Reelin(+/+) neurons beyond an unknown threshold resulted in partial rescue, with the formation of a correctly layered secondary cortex lying on top of an inverted mutant cortex. Therefore, the development of cortical layers in the correct order requires a minimal level of Reelin protein to be present although paradoxically, this is insufficient to prevent the simultaneous formation of inverted cortical layers in the same hemisphere.

Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2.

Many ion channels and transporters are regulated by ubiquitination mediated by the Nedd4 family of HECT-type ubiquitin ligases (E3s). These E3s commonly interact with substrates via their WW domains that bind to specific motifs in target proteins. However, not all potential targets of these E3s contain WW-binding motifs. Therefore, accessory proteins may mediate the interaction between Nedd4 family members and their targets. Here we report that the divalent metal ion transporter DMT1, the primary nonheme iron transporter in mammals, is regulated by ubiquitination mediated by the Nedd4 family member WWP2. DMT1 interacts with 2 WW domain-interacting proteins, Ndfip1 and Ndfip2, previously proposed to have roles in protein trafficking. This promotes DMT1 ubiquitination and degradation by WWP2. Consistent with these observations, Ndfip1(-/-) mice show increased DMT1 activity and a concomitant increase in hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. This novel mechanism of regulating iron homeostasis suggests that Ndfips and WWP2 may contribute to diseases involving aberrant iron transport.