西岡 朋生

Cytoskeletal remodeling drives morphological changes. Septin cytoskeleton assembles into hetero-oligomers. We previously demonstrated that late-phase long-term potentiation (L-LTP) induces smooth endoplasmic reticulum (sER) extension into dendritic spines via septin 3 (SEPT3), contributing to greater postsynaptic Ca2+ responses and enhanced activation of synaptically induced Ca2+ signaling. Sept3-/- mice exhibit a reduced number of sER-containing spines and show impaired long-term spatial/object memory despite normal short-term memory. Additionally, SEPT3 binds the motor protein myosin-Va (MYO5A) upon elevated Ca²⁺ concentrations, facilitating sER extension from the dendritic shaft into the spine. MYO5A localizes on the sER membrane, while SEPT3 remains at the spine base, accumulating on sER upon electroconvulsive stimulation (ECS). However, the mechanism underlying SEPT3's delocalization from the spine base and its cooperative role with MYO5A in sER extension remains unclear. In this study, we demonstrate that SEPT3 is phosphorylated in a stimulation-dependent manner. Phosphorylation at Thr211 releases SEPT3 from the spine base, enabling sER extension with constitutively active MYO5A mutant (MYO5A-CCtr). These findings provide molecular insight into the role of SEPT3 phosphorylation in regulating sER dynamics that sustain long-term spine activation.

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Discovery of novel post-translational modifications provides new insights into changes in protein function, localization, and stability. They are also key elements in understanding disease mechanisms and developing therapeutic strategies. We have previously reported that ubiquitin-like 3 (UBL3) serves as a novel post-translational modifier that is highly expressed in the cerebral cortex and hippocampus, in addition to various other organs, and that 60% of proteins contained in small extracellular vesicles (sEVs), including exosomes, are influenced by UBL3. In this study, we generated transgenic mice expressing biotinylated UBL3 in the forebrain under control of the alpha-CaMKII promoter (Ubl3Tg/+). Western blot analysis revealed that the expression of UBL3 in the cerebral cortex and hippocampus was 6- to 7-fold higher than that in the cerebellum. Therefore, we performed immunoprecipitation of protein extracts from the cerebral cortex of Ubl3+/+ and Ubl3Tg/+ mice using avidin beads to comprehensively discover UBL3 interacting proteins, identifying 35 new UBL3 interacting proteins. Nine proteins were annotated as extracellular exosomes. Gene Ontology (GO) analysis suggested a new relationship between sEVs and RNA metabolism in neurodegenerative diseases. We confirmed the association of endogenous UBL3 with the RNA-binding proteins FUS and HPRT1-both listed in the Neurodegenerative Diseases Variation Database (NDDVD)-and with LYPLA1, which is involved in Huntington's disease, using immunoprecipitation (IP)-western blotting analysis. These UBL3 interacting proteins will accelerate the continued elucidation of sEV research about proteins regulated by novel post-translational modifications by UBL3 in the brain.

Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named “Kinase-Associated Neural PHOspho-Signaling (KANPHOS).” This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp.

Clathrin-dependent endocytosis is a key process for secretory cells, in which molecules on the plasma membrane are both degraded and recycled in a stimulus-dependent manner. There are many reports showing that disruption of endocytosis is involved in the onset of various diseases. Recently, it has been reported that such disruption in pancreatic β-cells causes impaired insulin secretion and might be associated with the pathology of diabetes mellitus. Compared with exocytosis, there are few reports on the molecular mechanism of endocytosis in pancreatic β-cells. We previously reported that GDP-bound Rab27a regulates endocytosis through its GDP-dependent effectors after insulin secretion. In this study, we identified heat shock protein family A member 8 (HSPA8) as a novel interacting protein for GDP-bound Rab27a. HSPA8 directly bound GDP-bound Rab27a via the β2 region of its substrate binding domain (SBD). The β2 fragment was capable of inhibiting the interaction between HSPA8 and GDP-bound Rab27a, and suppressed glucose-induced clathrin-dependent endocytosis in pancreatic β-cells. The region also affected clathrin dynamics on purified clathrin-coated vesicles (CCVs). These results suggest that the interaction between GDP-bound Rab27a and HSPA8 regulates clathrin disassembly from CCVs and subsequent vesicle transport. The regulatory stages in endocytosis by HSPA8 differ from those for other GDP-bound Rab27a effectors. This study shows that GDP-bound Rab27a dominantly regulates each stage in glucose-induced endocytosis through its specific effectors in pancreatic β-cells.