| 研究実績の概要 |
To elucidate the molecular mechanism underlying the memory formation, we need to understand the regulatory mechanism of synaptic proteins of post-synaptic density (PSD). It is known that the PSD is enlarged after learning to enhance the synaptic transmission, and shrinks to maintain the homeostatic plasticity. Previously, we have reported a synaptic activity dependent organization of post-synaptic proteins via liquid-liquid phase separation (LLPS). Also, we found a short peptide (78 a.a.), Camk2n1, acts as the dissociation factor of PSD protein condensate. Thus, we hypothesized that Camk2n1 is the factor which regulates the homeostatic plasticity through balancing the hyperactivity that induced by the learning events and scale down the number of synapses to maintain the plasticity of entire neuronal network.
To test our hypothesis, in our first step, we want to observe the expression and localization of Camk2n1 in neuron. To do so, we have established the dissociated neuronal culture in this annual year. Also, we have constructed the CRISPR/Cas9-mediated knock-in (KI) GFP-tagged Camk2n1 and expressed it in neuron successfully. Next step, we will induce a chemical long-term potentiation to test if the expression of Camk2n1 would be increased or the localization would be changed after stimulation. Also, we are going to KI GFP-Camk2n1 in rat embryo, to observe Camk2n1 expression in living animal.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
In the beginning of this project, the major challenge is to observe endogenous Camk2n1 in neuron. The expression and localization of Camk2n1 have been observed with immunofluorescence, however, the antibodies are not really reliable. We chose to use a newly developed CRISPR/Cas9 KI system, Open Resource for the Application of Neuronal Genome Editing (ORANGE), to construct the endogenously GFP-tagged Camk2n1. At first we were not able to observe any signal, and we didn’t know it was due to low transfection efficiency with the ORANGE system or the expression of endogenous Camk2n1 was originally low at basal state. But by testing with different approaches to deliver the plasmid to neuronal culture, such as lipofection and electroporation, and enhancing the signal with immunofluorescence, we eventually confirmed the ORANGE system works well, and observe the GFP-tagged Camk2n1.
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| 今後の研究の推進方策 |
The synapses of neuron are the basic elements that enable information transferring in our brain to form memory, which is a critical ability for animal to survive. At the same time, the pathological disruptions in synapse structure are widely reported in the mental disorders and neurodegenerative diseases such as schizophrenia, dementia and Alzheimer’s disease. Therefore, understanding the regulatory mechanism of synaptic plasticity is important for the medical invention of memory-related mental disorders and neurodegenerative diseases, such as post-traumatic stress disorder (PTSD), depression and dementia.
So far, we have managed to observe the dissociation factor, Camk2n1, in neuron. We will use this to investigate the mechanism of synaptic down-regulation in the next step.
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