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.

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.

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.

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.

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.

Cell-type specific expression of constitutively-active Rheb promotes regeneration of bulbospinal respiratory axons following cervical SCI.

Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal-intrinsic axon growth capacity can promote regeneration of injured bulbospinal respiratory axons after SCI, but this strategy may need to be combined with other manipulations to achieve reconnection of damaged neural circuitry and ultimately recovery of diaphragm function.