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.

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 [...].

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.

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.

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.

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.

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.

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.

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.

Methionine sulfoxide reductase A (MsrA) mediates the ubiquitination of 14-3-3 protein isotypes in brain.

The methionine sulfoxide reductase (Msr) system is known for its function in reducing protein-methionine sulfoxide to methionine. Recently, we showed that one member of the Msr system, MsrA, is involved in the ubiquitination-like process in Archaea. Here, the mammalian MsrA is demonstrated to mediate the ubiquitination of the 14-3-3 zeta protein and to promote the binding of 14-3-3 proteins to alpha synuclein in brain. MsrA was also found to enhance the ubiquitination and phosphorylation of Ser129 of alpha synuclein in brain. Furthermore, we demonstrate that, similarly to the archaeal MsrA, the mammalian MsrA can compete for capturing ubiquitin using the same active site it contains for methionine sulfoxide binding. Based on our previous observations showing that MsrA knockout mice have elevated expression levels of dopamine and 14-3-3 zeta and our current data, we propose that MsrA-dependent 14-3-3 zeta ubiquitination affects the regulation of alpha synuclein degradation and dopamine synthesis in the brain.

Neurodevelopmental Genetic Diseases Associated With Microdeletions and Microduplications of Chromosome 17p13.3.

Chromosome 17p13.3 is a region of genomic instability that is linked to different rare neurodevelopmental genetic diseases, depending on whether a deletion or duplication of the region has occurred. Chromosome microdeletions within 17p13.3 can result in either isolated lissencephaly sequence (ILS) or Miller-Dieker syndrome (MDS). Both conditions are associated with a smooth cerebral cortex, or lissencephaly, which leads to developmental delay, intellectual disability, and seizures. However, patients with MDS have larger deletions than patients with ILS, resulting in additional symptoms such as poor muscle tone, congenital anomalies, abnormal spasticity, and craniofacial dysmorphisms. In contrast to microdeletions in 17p13.3, recent studies have attracted considerable attention to a condition known as a 17p13.3 microduplication syndrome. Depending on the genes involved in their microduplication, patients with 17p13.3 microduplication syndrome may be categorized into either class I or class II. Individuals in class I have microduplications of the YWHAE gene encoding 14-3-3ε, as well as other genes in the region. However, the PAFAH1B1 gene encoding LIS1 is never duplicated in these patients. Class I microduplications generally result in learning disabilities, autism, and developmental delays, among other disorders. Individuals in class II always have microduplications of the PAFAH1B1 gene, which may include YWHAE and other genetic microduplications. Class II microduplications generally result in smaller body size, developmental delays, microcephaly, and other brain malformations. Here, we review the phenotypes associated with copy number variations (CNVs) of chromosome 17p13.3 and detail their developmental connection to particular microdeletions or microduplications. We also focus on existing single and double knockout mouse models that have been used to study human phenotypes, since the highly limited number of patients makes a study of these conditions difficult in humans. These models are also crucial for the study of brain development at a mechanistic level since this cannot be accomplished in humans. Finally, we emphasize the usefulness of the CRISPR/Cas9 system and next generation sequencing in the study of neurodevelopmental diseases.

14-3-3 Proteins in Brain Development: Neurogenesis, Neuronal Migration and Neuromorphogenesis.

The 14-3-3 proteins are a family of highly conserved, multifunctional proteins that are highly expressed in the brain during development. Cumulatively, the seven 14-3-3 isoforms make up approximately 1% of total soluble brain protein. Over the last decade, evidence has accumulated implicating the importance of the 14-3-3 protein family in the development of the nervous system, in particular cortical development, and have more recently been recognized as key regulators in a number of neurodevelopmental processes. In this review we will discuss the known roles of each 14-3-3 isoform in the development of the cortex, their relation to human neurodevelopmental disorders, as well as the challenges and questions that are left to be answered. In particular, we focus on the 14-3-3 isoforms and their involvement in the three key stages of cortical development; neurogenesis and differentiation, neuronal migration and neuromorphogenesis and synaptogenesis.

Complete ablation of the 14-3-3epsilon protein results in multiple defects in neuropsychiatric behaviors.

Previous studies show that mice with Ywhae deficiency show abnormalities in brain development including defects in neuronal migration of post-mitotic pyramidal neurons as well as neuronal differentiation and proliferation in neuronal progenitor cells. Also, our previous research indicated that the Ywhae knockout mice show moderate defects in working memory and anxiety-like behavior. This previous work was performed using heterozygous mutant mice. Here we performed behavioral analyses using homozygous Ywhae knockout mice and found that the homozygous Ywhae knockout mice have increased locomotor activity, decreased working memory, and increased sociability. Taken together with the results obtained from the previous pathophysiological analyses in the Ywhae knockout mice, the Ywhae knockout mouse is useful for pathophysiological analyses of neuropsychiatric disorders caused by defects during neurodevelopment.

Overexpression of the 14-3-3gamma protein in embryonic mice results in neuronal migration delay in the developing cerebral cortex.

The 14-3-3 protein family is a group of multifunctional proteins that are highly expressed in the brain; however, their functions in brain development are largely unknown. Williams Syndrome is a neurodevelopmental disorder caused by a deletion in the 7q11.23 chromosome locus, including the gene encoding 14-3-3gamma, resulting in developmental delay, intellectual disabilities and epilepsy. We have previously shown that knocking down the 14-3-3gamma protein in utero in mice results in delays in neuronal migration of pyramidal neurons in the cortex. Importantly, there is a reciprocal duplication syndrome to Williams Syndrome where the 7q11.23 locus is duplicated, resulting in epilepsy and intellectual disabilities. Thus, the deletion or the duplication of the 7q11.23 chromosome locus results in epilepsy. Taken together with the fact that defects in neuronal migration are one of main causes for epilepsy, we analyzed if the overexpression of 14-3-3gamma causes neuronal migration defects. In this work, we found that the overexpression of 14-3-3gamma in utero in the developing mouse cortex results in delays in pyramidal neuron migration, similar to what was previously observed when 14-3-3gamma was knocked down. These results, in conjunction with our previous research, indicate that a balance of 14-3-3gamma expression is required during cortical development to prevent delays in neuronal migration. This work provides clear evidence as to the involvement of 14-3-3gamma in neurodevelopmental disorders and how a disruption in 14-3-3gamma expression may contribute to the neurodevelopmental disorders that manifest when the 7q11.23 locus is altered.

Deficiency of 14-3-3ε and 14-3-3ζ by the Wnt1 promoter-driven Cre recombinase results in pigmentation defects.

BACKGROUND: The seven 14-3-3 protein isoforms bind to numerous proteins and are involved in a wide variety of cellular events, including the cell cycle, cell division, apoptosis and cancer. We previously found the importance of 14-3-3 proteins in neuronal migration of pyramidal neurons in the developing cortex. Here, we test the function of 14-3-3 proteins in the development of neural crest cells in vivo using mouse genetic approaches. RESULTS: We found that 14-3-3 proteins are important for the development of neural crest cells, in particular for the pigmentation of the fur on the ventral region of mice. CONCLUSIONS: Our data obtained from the 14-3-3ε/14-3-3ζ/Wnt1-Cre mice strongly indicate the importance of 14-3-3 proteins in the development of melanocyte lineages.

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.