The cytoplasmic localization of ADNP through 14-3-3 promotes sex-dependent neuronal morphogenesis, cortical connectivity, and calcium signaling.

Defective neuritogenesis is a contributing pathogenic mechanism underlying a variety of neurodevelopmental disorders. Single gene mutations in activity-dependent neuroprotective protein (ADNP) are the most frequent among autism spectrum disorders (ASDs) leading to the ADNP syndrome. Previous studies showed that during neuritogenesis, Adnp localizes to the cytoplasm/neurites, and Adnp knockdown inhibits neuritogenesis in culture. Here, we hypothesized that Adnp is localized in the cytoplasm during neurite formation and that this process is mediated by 14-3-3. Indeed, applying the 14-3-3 inhibitor, difopein, blocked Adnp cytoplasmic localization. Furthermore, co-immunoprecipitations showed that Adnp bound 14-3-3 proteins and proteomic analysis identified several potential phosphorylation-dependent Adnp/14-3-3 binding sites. We further discovered that knockdown of Adnp using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex led to previously unreported changes in neurite formation beginning at P0. Defects were sustained throughout development, the most notable included increased basal dendrite number and axon length. Paralleling the observed morphological aberrations, ex vivo calcium imaging revealed that Adnp deficient neurons had greater and more frequent spontaneous calcium influx in female mice. GRAPHIC, a novel synaptic tracing technology substantiated this finding, revealing increased interhemispheric connectivity between female Adnp deficient layer 2/3 pyramidal neurons. We conclude that Adnp is localized to the cytoplasm by 14-3-3 proteins, where it regulates neurite formation, maturation, and functional cortical connectivity significantly building on our current understanding of Adnp function and the etiology of ADNP syndrome.

KIFC1 Regulates the Trajectory of Neuronal Migration.

During neuronal migration, forces generated by cytoplasmic dynein yank on microtubules extending from the centrosome into the leading process and move the nucleus along microtubules that extend behind the centrosome. Scaffolds, such as radial glia, guide neuronal migration outward from the ventricles, but little is known about the internal machinery that ensures that the soma migrates along its proper path rather than moving backward or off the path. Here we report that depletion of KIFC1, a minus-end-directed kinesin called HSET in humans, causes neurons to migrate off their appropriate path, suggesting that this molecular motor is what ensures fidelity of the trajectory of migration. For these studies, we used rat migratory neurons in vitro and developing mouse brain in vivo, together with RNA interference and ectopic expression of mutant forms of KIFC1. We found that crosslinking of microtubules into a nonsliding mode by KIFC1 is necessary for dynein-driven forces to achieve sufficient traction to thrust the soma forward. Asymmetric bouts of microtubule sliding driven by KIFC1 thereby enable the soma to tilt in one direction or another, thus providing midcourse corrections that keep the neuron on its appropriate trajectory. KIFC1-driven sliding of microtubules further assists neurons in remaining on their appropriate path by allowing the nucleus to rotate directionally as it moves, which is consistent with how we found that KIFC1 contributes to interkinetic nuclear migration at an earlier stage of neuronal development.SIGNIFICANCE STATEMENT Resolving the mechanisms of neuronal migration is medically important because many developmental disorders of the brain involve flaws in neuronal migration and because deployment of newly born neurons may be important in the adult for cognition and memory. Drugs that inhibit KIFC1 are candidates for chemotherapy and therefore should be used with caution if they are allowed to enter the brain.

Glutathione S-transferase Pi (Gstp) proteins regulate neuritogenesis in the developing cerebral cortex.

GSTP proteins are metabolic enzymes involved in the removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, whereas there is only one GSTP in humans. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown (KD) caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation (IUE) to knock down Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared with the control. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division.

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.

Rpsa Signaling Regulates Cortical Neuronal Morphogenesis via Its Ligand, PEDF, and Plasma Membrane Interaction Partner, Itga6.

Neuromorphological defects underlie neurodevelopmental disorders and functional defects. We identified a function for Rpsa in regulating neuromorphogenesis using in utero electroporation to knockdown Rpsa, resulting in apical dendrite misorientation, fewer/shorter extensions, and decreased spine density with altered spine morphology in upper neuronal layers and decreased arborization in upper/lower cortical layers. Rpsa knockdown disrupts multiple aspects of cortical development, including radial glial cell fiber morphology and neuronal layering. We investigated Rpsa's ligand, PEDF, and interacting partner on the plasma membrane, Itga6. Rpsa, PEDF, and Itga6 knockdown cause similar phenotypes, with Rpsa and Itga6 overexpression rescuing morphological defects in PEDF-deficient neurons in vivo. Additionally, Itga6 overexpression increases and stabilizes Rpsa expression on the plasma membrane. GCaMP6s was used to functionally analyze Rpsa knockdown via ex vivo calcium imaging. Rpsa-deficient neurons showed less fluctuation in fluorescence intensity, suggesting defective subthreshold calcium signaling. The Serpinf1 gene coding for PEDF is localized at chromosome 17p13.3, which is deleted in patients with the neurodevelopmental disorder Miller-Dieker syndrome. Our study identifies a role for Rpsa in early cortical development and for PEDF-Rpsa-Itga6 signaling in neuromorphogenesis, thus implicating these molecules in the etiology of neurodevelopmental disorders like Miller-Dieker syndrome and identifying them as potential therapeutics.

Protein kinases: master regulators of neuritogenesis and therapeutic targets for axon regeneration.

Proper neurite formation is essential for appropriate neuronal morphology to develop and defects at this early foundational stage have serious implications for overall neuronal function. Neuritogenesis is tightly regulated by various signaling mechanisms that control the timing and placement of neurite initiation, as well as the various processes necessary for neurite elongation to occur. Kinases are integral components of these regulatory pathways that control the activation and inactivation of their targets. This review provides a comprehensive summary of the kinases that are notably involved in regulating neurite formation, which is a complex process that involves cytoskeletal rearrangements, addition of plasma membrane to increase neuronal surface area, coupling of cytoskeleton/plasma membrane, metabolic regulation, and regulation of neuronal differentiation. Since kinases are key regulators of these functions during neuromorphogenesis, they have high potential for use as therapeutic targets for axon regeneration after injury or disease where neurite formation is disrupted.

Regulation of neuronal morphogenesis by 14-3-3epsilon (Ywhae) via the microtubule binding protein, doublecortin.

17p13.3 microduplication syndrome is a newly identified genetic disorder characterized by duplications in the 17p13.3 chromosome locus, resulting in a variety of disorders including autism spectrum disorder (ASD). Importantly, a minimum duplication region has been defined, and this region exclusively contains the gene encoding 14-3-3ε. Furthermore, duplication of this minimum region is strongly associated with the appearance of ASD in human patients, thus implicating the overexpression of 14-3-3ε in ASD. Using in vitro and in vivo techniques, we have found that 14-3-3ε binds to the microtubule binding protein doublecortin preventing its degradation. We also found that 14-3-3ε overexpression disrupts neurite formation by preventing the invasion of microtubules into primitive neurites, which can be rescued by the knockdown of doublecortin. To analyse the function of 14-3-3ε in neurite formation, we used 14-3-3ε flox mice and found that 14-3-3ε deficiency results in an increase in neurite formation. Our findings provide the first evidence of cellular pathology in 17p13.3 microduplication syndrome.

14-3-3ε and ζ regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain.

During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of ß-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades.

14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for Miller-Dieker syndrome.

Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3epsilon (YWHAE), one of a family of ubiquitous phosphoserine/threonine-binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3epsilon results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3epsilon in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.

Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation.

Pyramidal neurons are a major cell type of the forebrain, consisting of a pyramidally shaped soma with axonal and apicobasal dendritic processes. It is poorly understood how the neuronal soma develops its pyramidal morphology, while generating neurites of the proper shape and orientation. Here, we discovered that the spherical somata of immature neurite-less neurons possess a circumferential wreath-like network of septin filaments, which promotes neuritogenesis by balancing the protrusive activity of lamellipodia and filopodia. In embryonic rat hippocampal and mouse cortical neurons, the septin wreath network consists of curvilinear filaments that contain septins 5, 7, and 11 (Sept5/7/11). The Sept5/7/11 wreath network demarcates a zone of myosin II enrichment and Arp2/3 diminution at the base of filopodial actin bundles. In Sept7-depleted neurons, cell bodies are enlarged with hyperextended lamellae and abnormally shaped neurites that originate from lamellipodia. This phenotype is accompanied by diminished myosin II and filopodia lifetimes and increased Arp2/3 and lamellipodial activity. Inhibition of Arp2/3 rescues soma and neurite phenotypes, indicating that the septin wreath network suppresses the extension of lamellipodia, facilitating the formation of neurites from the filopodia of a consolidated soma. We show that this septin function is critical for developing a pyramidally shaped soma with properly distributed and oriented dendrites in cultured rat hippocampal neurons and in vivo in mouse perinatal cortical neurons. Therefore, the somatic septin cytoskeleton provides a key morphogenetic mechanism for neuritogenesis and the development of pyramidal neurons.

Nuak kinase signaling in development and disease of the central nervous system.

Protein kinases represent important signaling hubs for a variety of biological functions. Many kinases are traditionally studied for their roles in cancer cell biology, but recent advances in neuroscience research show repurposed kinase function to be important for nervous system development and function. Two members of the AMP-activated protein kinase (AMPK) related family, NUAK1 and NUAK2, have drawn attention in neuroscience due to their mutations in autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), schizophrenia, and intellectual disability (ID). Furthermore, Nuak kinases have also been implicated in tauopathy and other disorders of aging. This review highlights what is known about the Nuak kinases in nervous system development and disease and explores the possibility of Nuak kinases as targets for therapeutic innovation.

Correction: Liu et al. Responsible Genes for Neuronal Migration in the Chromosome 17p13.3: Beyond Pafah1b1(Lis1), Crk and Ywhae(14-3-3ε). Brain Sci. 2022, 12, 56.

The authors wish to correct the following error in this paper [...].

High-throughput kinase inhibitor screening reveals roles for Aurora and Nuak kinases in neurite initiation and dendritic branching.

Kinases are essential regulators of a variety of cellular signaling processes, including neurite formation-a foundational step in neurodevelopment. Aberrant axonal sprouting and failed regeneration of injured axons are associated with conditions like traumatic injury, neurodegenerative disease, and seizures. Investigating the mechanisms underlying neurite formation will allow for identification of potential therapeutics. We used a kinase inhibitor library to screen 493 kinase inhibitors and observed that 45% impacted neuritogenesis in Neuro2a (N-2a) cells. Based on the screening, we further investigated the roles of Aurora kinases A, B, and C and Nuak kinases 1 and 2. The roles of Aurora and Nuak kinases have not been thoroughly studied in the nervous system. Inhibition or overexpression of Aurora and Nuak kinases in primary cortical neurons resulted in various neuromorphological defects, with Aurora A regulating neurite initiation, Aurora B and C regulating neurite initiation and elongation, all Aurora kinases regulating arborization, and all Nuak kinases regulating neurite initiation and elongation and arborization. Our high-throughput screening and analysis of Aurora and Nuak kinases revealed their functions and may contribute to the identification of therapeutics.

Responsible Genes for Neuronal Migration in the Chromosome 17p13.3: Beyond Pafah1b1(Lis1), Crk and Ywhae(14-3-3ε).

The 17p13.3 chromosome region is often deleted or duplicated in humans, resulting in severe neurodevelopmental disorders such as Miller-Dieker syndrome (MDS) and 17p13.3 duplication syndrome. Lissencephaly can also be caused by gene mutations or deletions of a small piece of the 17p13.3 region, including a single gene or a few genes. PAFAH1B1 gene, coding for LIS1 protein, is a responsible gene for lissencephaly and MDS and regulates neuronal migration by controlling microtubules (MTs) and cargo transport along MTs via dynein. CRK is a downstream regulator of the reelin signaling pathways and regulates neuronal migration. YWHAE, coding for 14-3-3ε, is also responsible for MDS and regulates neuronal migration by binding to LIS1-interacting protein, NDEL1. Although these three proteins are known to be responsible for neuronal migration defects in MDS, there are 23 other genes in the MDS critical region on chromosome 17p13.3, and little is known about their functions in neurodevelopment, especially in neuronal migration. This review will summarize the recent progress on the functions of LIS1, CRK, and 14-3-3ε and describe the recent findings of other molecules in the MDS critical regions in neuronal migration.

TNFR2/14-3-3ε signaling complex instructs macrophage plasticity in inflammation and autoimmunity.

TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPß activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.

Identification of YWHAE, a gene encoding 14-3-3epsilon, as a possible susceptibility gene for schizophrenia.

Schizophrenia is a complex mental disorder with a fairly high degree of heritability. Although the causes of schizophrenia remain unclear, it is now widely accepted that it is a neurodevelopmental and neurodegenerative disorder involving disconnectivity and disorder of the synapses. Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene involved in neurodevelopment, including maturation of the cerebral cortex. To identify other susceptibility genes for schizophrenia, we screened for DISC1-interacting molecules [NudE-like (NUDEL), Lissencephaly-1 (LIS1), 14-3-3epsilon (YWHAE), growth factor receptor bound protein 2 (GRB2) and Kinesin family 5A of Kinesen1 (KIF5A)], assessing a total of 25 tagging single-nucleotide polymorphisms (SNPs) in a Japanese population. We identified a YWHAE SNP (rs28365859) that showed a highly significant difference between case and control samples, with higher minor allele frequencies in controls (P(allele) = 1.01 x 10(-5) and P(genotype) = 4.08 x 10(-5) in 1429 cases and 1728 controls). Both messenger RNA transcription and protein expression of 14-3-3epsilon were also increased in the lymphocytes of healthy control subjects harboring heterozygous and homozygous minor alleles compared with homozygous major allele subjects. To further investigate a potential role for YWHAE in schizophrenia, we studied Ywhae(+/-) mice in which the level of 14-3-3epsilon protein is reduced to 50% of that in wild-type littermates. These mice displayed weak defects in working memory in the eight-arm radial maze and moderately enhanced anxiety-like behavior in the elevated plus-maze. Our results suggest that YWHAE is a possible susceptibility gene that functions protectively in schizophrenia.

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment.

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality.

Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.