Neuron Tan Chao et al. A small molecule protein developed based on the zinc finger domain of RIM protein and calcium channel subunit β4 to rebuild the core function of the active area in the synapse

Editor-in-Chief | XiThe function of the brain is highly dependent on precisely regulated synaptic transmission
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The active zone is a molecular machine that controls the secretion of presynaptic neurotransmitters
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The active region consists of multiple protein families, including RIM, ELKS, Munc13, RIM-BP, Bassoon/Piccolo and Liprin-α (see Figure 1)
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These proteins have molecular weights ranging from 120 to 420 kDa, and they interact to form complex protein networks
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The main function of the active zone is to achieve vesicle docking with the presynaptic membrane (vesicle docking), increase the fusion ability of vesicles (vesicle priming), and couple calcium ions to guide vesicle fusion to complete neurotransmitter release
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Previous studies have found that the protein network of the active region "drives the whole body", that is, knocking out any protein in the active region alone will cause defects in the function of the active region
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Therefore, it is difficult for researchers to determine which proteins play a central role in the control of transmitter release in the active region
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A new research idea is to search for the core constituent elements of active regions by reconstructing their functions, but so far it has not been realized
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Figure 1: Schematic diagram of active zone structure in synapses On February 16, 2022, Dr.
Tan Chao from Pascal Kaeser's laboratory at Harvard Medical School and his colleagues published a paper entitled Rebuilding essential active zone functions within a synapse in Neuron.
's thesis
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In this paper, the authors reconstruct the function of the active region by disrupting the protein network of the active region
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First, the authors disrupted the active zone by knocking out RIM and ELKS in primary hippocampal neurons
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They observed that knockdown of RIM and ELKS in neurons resulted in an almost complete loss of RIM1, ELKS2, and Munc13-1 proteins in the active region, a substantial reduction in Bassoon and RIM-BP2 proteins, and a partial loss of the CaV2.
1 channel protein
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In addition, knockdown of RIM and ELKS in neurons abolished the docking of vesicles to the presynaptic membrane, fusion-capable vesicles decreased by approximately 70%, and the coupling of vesicles to calcium ions was severely impaired.
These results Indicates that the active region protein network has been disrupted
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Next, the authors set out to reconstruct the function of the active region, first by re-expressing RIM or ELKS
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The authors found that the newly expressed ELKS2 protein could not localize to the active region, but the newly expressed RIM1 protein could localize to the active region
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Moreover, the newly expressed RIM1 protein also restored the expression levels of other proteins in the active region, including Munc13-1, RIM-BP2, Bassoon and CaV2.
1, so RIM1 may be a key protein controlling the protein network in the active region
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In order to elucidate the core constituent elements of the active region, the authors hoped to use the smallest possible protein domains to reconstruct the function of the active region.
To this end, the authors constructed multiple mutations of the RIM1 domain
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The authors found that a single zinc finger domain (Zn) in RIM1 can localize to the presynaptic vesicle pool
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Notably, the zinc finger domain in RIM1 recruits Munc13-1 protein to the presynaptic vesicle pool as well, and their interaction increases the fusion capacity of the vesicles
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However, the docking of the vesicles to the presynaptic membrane was not altered
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This is a very interesting phenomenon because conventional wisdom holds that vesicles first dock with the presynaptic membrane, and then the fusion capacity of the vesicles is enhanced (see Figure 2, image from Ref.
2), or that both processes occur simultaneously ( Reference 3)
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The results of this study challenge the traditional "vesicle docking first, then enhance vesicle fusion capacity" model.
Instead, this study shows that increasing vesicle fusion capacity and vesicle docking with the presynaptic membrane are separate processes , the order in which these two processes occur can be changed
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Figure 2: Schematic illustration of the process of vesicle release and recycling.
Following this, the authors aimed to restore vesicle docking to the presynaptic membrane by targeting the RIM1 zinc finger domains to CaV2 channels, allowing fusion-capable vesicles to respond to the action potential.
Induced calcium influx completes vesicle fusion, thereby promoting neurotransmitter release
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The authors linked the RIM1 zinc finger domain directly to the calcium channel subunit CaVβ4, which they named β4-Zn
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The authors found that β4-Zn could localize to the active region and increased the expression levels of Munc13-1 and CaV2 proteins in the active region
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Moreover, β4-Zn completely rebuilt the function of the active zone, including the docking of vesicles with the presynaptic membrane, increasing the fusion ability of vesicles, and coupling calcium ions to guide vesicle fusion.

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Further studies found that the function of β4-Zn mainly depended on its recruitment to Munc13-1 and its binding to Munc13-1 (see Figure 3), and did not recruit other active region proteins, such as Bassoon or RIM-BP2 proteins
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Figure 3: Schematic representation of the reconstructed active zone Finally, the authors supplemented their experiments to establish active zones by transfection in non-neuronal cells
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Co-transfection of β4-Zn, Munc13-1, CaV2.
1 and a2d1 formed a protein complex on HEK293T cell membranes that closely resembled the docking complex at the presynaptic membrane in neurons
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To test whether this complex could tether vesicles to the membrane, the authors fused the RIM1 zinc finger domain to the outer layer of the transmembrane region of the mitochondrial protein Tom20 in neurons and named it mitoC-Zn
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The authors found that mitoC-Zn targets mitochondria, while mitoC-Zn recruits Munc13-1 to the mitochondrial surface and tethers vesicles to the mitochondrial surface
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Overall, the authors achieved intrasynaptic vesicle fusion by targeting the RIM zinc finger protein domain, a small protein domain capable of recruiting Munc13-1, to presynaptic calcium channels Fast and precise release of neurotransmitters
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This work reveals that increasing the fusion capacity of vesicles and the docking of vesicles to the presynaptic membrane are separable processes, and that vesicles far from the active zone can be activated for fusion
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This study also shows that β4-Zn skips the complex active region protein network to rebuild the function of the active region, and is an artificial simplified version of the active region.
The small molecular weight β4-Zn (80 kDa) can be used for AAV virus packaging , has potential application value for improving disease-induced synaptic transmission defects
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Original link: https://doi.
org/10.
1016/j.
neuron.
2022.
01.
026 Publisher: Eleven References 1.
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, Rebuilding essential active zone functions within a synapse, Neuron (2022), https: //doi.
org/10.
1016/j.
neuron.
2022.
01.
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, Sudhof, TC, Rhee, JS, Brose, N.
, and Cooper, BH (2014).
The morphological and molecular nature of Synaptic vesicle priming at presynaptic active zones.
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