Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

Abstract

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

“…Sprague Dawley rat hippocampal and cortical neurons were isolated at E18 (Envigo). Mouse hippocampal neurons from the syt7 floxed mouse strain Syt7-TM1C-EM4-B6N (Codner et al, 2018) and syt7 tm1Nan (Chakrabarti et al, 2003) were isolated at P0 and prepared using a procedure previously described in (Vevea & Chapman, 2020). Briefly, hippocampal neurons were dissected, trypsinized (Corning; 25-053-CI), triturated, and plated on glass coverslips (Warner instruments; 64-0734 (CS-18R17)) coated with poly-D-lysine (Thermofisher; ICN10269491) and EHS laminin (Thermofisher; 23017015).…”

“…One was our previously modified lentivirus backbone of choice derived from FUGW (Vevea & Chapman, 2020), and the other was based off pEF-GFP, excising the GFP and substituting our own various inserts (pEF-GFP was a gift from Connie Cepko (Addgene plasmid # 11154 ; http://n2t.net/addgene:11154 ; RRID:Addgene_11154)) (Matsuda & Cepko, 2004). The low affinity glutamate sensor iGluSnFR S72A was PCR amplified from the pAAV.hSynapsin.SF-iGluSnFR.S72A which was a gift from Loren Looger (Addgene plasmid # 106176; https://www.addgene.org/106176/; RRID:Addgene_106176)) (Marvin et al, 2018), and subcloned into our lentivirus transfer plasmid (CamKII promoter) along with the addition of membrane trafficking motifs to promote plasma membrane localization as done previously for the original iGluSnFR variant (Vevea & Chapman, 2020). The vGlut1-pHluorin construct used here was subcloned into our modified FUGW transfer vector (hSynapsin promoter) from the original vGlut1-pHluorin construct (Voglmaier et al, 2006).…”

Metabotropic glutamate receptor 5 regulates synaptic plasticity in a chronic migraine rat model through the PKC/NR2B signal

“…Its expression level can indirectly reflect the integrity and functional state of synaptic structure and is a specific marker of synaptic plasticity [42]. Syt-1 is a calcium-binding protein that dominates the release of calcium-dependent transmitters, participates in the discharge and transport of synaptic vesicles, affects synaptic function and structure, and is a marker of synaptic plasticity [43]. Similar to previous studies, our study found that the expression of these synaptic structures and function related proteins was significantly increased in CM, and the administration of MPEP downregulated their expression and regulated synaptic plasticity.…”

Metabotropic Glutamate Receptor 5 Regulates Synaptic Plasticity in a Chronic Migraine Rat Model Through the PKC/NR2B Signal

“…Its expression level can indirectly re ect the integrity and functional state of synaptic structure, and is a speci c marker of synaptic plasticity [37]. Syt-1 is a calcium-binding protein that dominates the release of calcium-dependent transmitters, participates in the discharge and transport of synaptic vesicles, affects synaptic function and structure, and is a marker of synaptic plasticity [38]. Similar to previous studies, our study found that the expression of these synaptic structure and function related proteins was signi cantly increased in CM, and the administration of MPEP downregulated their expression and regulated synaptic plasticity.…”