Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Abstract

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.

Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse

“…This number is strikingly close to the average number of synaptic vesicles found in synaptic boutons of rat hippocampal neurons (195) [52]. Given recent studies estimating that the half-life of synaptic vesicles at presynaptic sites is below 30 hr [2], our results indicate that a typical synapse can be fully supplied with new synaptic vesicles approximately at the same rate that older synaptic vesicles are targeted for degradation.…”

“…Neurons were imaged between 14-17 DIV, a time point when these are synaptically connected and fire spontaneously [2]. Imaging of live neurons was performed in Tyrode's buffer (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 25 mM HEPES, 30 mM D-glucose, buffered to pH 7.4 at 37 C), using a PerkinElmer UltraVIEW VOX Spinning Disk Confocal with a Nikon Eclipse Ti Inverted Microscope in an environmental chamber at 37 C. This system is equipped with a Plan Apochromat Lambda 60x 1.40 NA and an Apochromat 100x 1.49 NA oil-immersion objectives, and a PhotoKinesis accessory for photobleaching.…”

“…After fusion, synaptic vesicles are endocytosed, refilled with neurotransmitter, and may undergo another round of fusion. Synaptic vesicles are estimated to recycle 200 times and remain in use for $18 hr, after which they are targeted for degradation [2]. CNS neurons can establish thousands of synaptic contacts en passant along their extensive axonal arbor [3].…”

Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

“…mediating the activity-dependent degradation of old and/or dysfunctional SV proteins. This hypothesis, to be further explored in future studies, aligns with previous research indicating that SV proteins become increasingly dysfunctional as they age and undergo multiple cycles of exo/endocytosis (Truckenbrodt et al 2018;Fernandes et al 2014).…”

Misfolded amyloid β-42 induced impairment of the endosomal-lysosomal pathway revealed by real-time optical monitoring

“…Specifically, we used CypHer5E, a commercially available pHsensitive fluorescent probe, which exhibits maximal emission when present in an acidic environment [37]. In previous studies this fluorescent moiety has been used to label antibodies for reporting receptor internalisation [38] and to tag vesicle proteins for assaying exo-endocytic recycling at synaptic terminals [39][40][41].…”