Biology subjects

Bennison, S. A.

Publications and source records attributed to Bennison, S. A..

4 recordsLinked to original sources

bioRxiv2020

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.

neuroscience↗

bioRxiv2020

14-3-3 shuttles Activity-dependent neuroprotective protein to the cytoplasm to promote appropriate neuronal morphogenesis, cortical connectivity and calcium signaling

Neurite formation is the earliest stage of neuronal morphogenesis, where primitive dendrites and the primitive axon emerge from a spherical neuron and begin to elongate. Defective neuritogenesis is a contributing pathogenic mechanism behind a variety of neurodevelopmental disorders. Activity-dependent neuroprotective protein (Adnp) is essential to embryonic and postnatal brain development, and mutations in ADNP are among the most frequent underlying autism spectrum disorder (ASD). We found that knockdown of Adnp in vitro and in vivo in mouse layer 2/3 pyramidal neurons leads to increased neurite initiation and defective neurite elongation, suggesting that Adnp has distinct roles in each. In vivo analysis revealed that deficits begin at P0 and are sustained throughout development, the most notable of which include increased neurite stabilization, disrupted angle of the apical dendrite, increased basal dendrite number, and increased axon length. Because small changes in neuronal morphology can have large-scale effects on neuronal function and connectivity, we performed ex vivo calcium imaging to assess spontaneous function of layer 2/3 pyramidal neurons deficient in Adnp. This revealed that Adnp deficient neurons had a greater spontaneous calcium influx and a higher proportion of cells firing action potentials. Next, we utilized GRAPHIC, a novel synaptic tracing technology, to assess interhemispheric cortical connectivity. We found increased interhemispheric excitatory connectivity between Adnp deficient layer 2/3 pyramidal neurons. Because Adnp is a multifunctional protein with both transcription factor and cytoskeletal activity, we performed localization analysis of Adnp as neurons underwent neurite formation to probe the mechanism of our morphological defects. We found that Adnp is shuttled from the nucleus to the cytoplasm upon differentiation and this shuttling can be blocked via application of a global 14-3-3 inhibitor, difopein. Furthermore, we found that Adnp binds nuclear-cytoplasmic shuttle 14-3-3{varepsilon}. We conclude that Adnp is shuttled from the nucleus to the cytoplasm by 14-3-3{varepsilon}, where it regulates neuronal morphology, maturation, cortical connectivity, and calcium signaling.

neuroscience↗

bioRxiv2020

Glutathione S-transferase Pi (Gstp) Proteins Regulate Neuritogenesis in the Developing Cerebral Cortex

GSTP proteins are metabolic enzymes involved in 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, while there is only one GSTP in humans. By 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 caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation to knockdown 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 to the control, but these defects were ameliorated. Overexpression of Gstp 1 or 2 resulted in changes in neurite length, suggesting a role in neurite elongation. 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. Furthermore, the overexpression experiments suggest different functions of Gstp1 and 2 in neurite elongation. Since previous studies have shown the potential implication of Gstp in Autism Spectrum Disorder, our findings will attract more clinical interests in Gstp proteins in neurodevelopmental disorders. SignificanceNeurite formation, including neurite initiation and elongation, is the first step of generating polarized neuronal morphology in developing neurons, and thus is essential for establishing a neuronal network. Therefore, it is crucial to understand the mechanisms of neurite formation. Limited studies have been performed to clarify the mechanisms of neurite formation, especially neurite initiation. In this present study, we report a novel, essential role of Gstp in neurite initiation in mouse cortical neurons in vitro and in vivo. We also found that Gstp regulates neurite formation via JNK signaling pathways. 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.

neuroscience↗

bioRxiv2020

PEDF-Rpsa-Itga6 signaling regulates cortical neuronal morphogenesis

Neuromorphological defects underlie neurodevelopmental disorders and functional defects. We identified a function for ribosomal protein SA (Rpsa) in regulating neuromorphogenesis using in utero electroporation to knockdown Rpsa, which results in apical dendrite misorientation, fewer/shorter extensions with decreased arborization, and decreased spine density with altered spine morphology. We investigated Rpsas ligand, pigment epithelium-derived factor (PEDF), and interacting partner on the plasma membrane, Integrin subunit 6 (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 by preventing ubiquitination of Rpsa. GCaMP6s was used to functionally analyze Rpsa knockdown via ex vivo calcium imaging. Rpsa deficient neurons showed less fluctuation in fluorescence intensity, suggesting defective sub-threshold calcium signaling. Our study identifies a role for PEDF-Rpsa-Itga6 signaling in neuromorphogenesis, thus implicating these molecules in the etiology of neurodevelopmental disorders and identifying them as potential therapeutic candidates.

neuroscience↗