Abstract
Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial.
Highlights
Chemical synapses contain multitudes of proteins, some of which play direct roles in synaptic transmission, whereas others regulate synaptic function or serve as structural scaffolds
To measure metabolic turnover rates of synaptic proteins we used dynamic SILAC (Stable Isotope Labeling with Amino acids in Cell culture) and mass spectrometry (MS) [25,26,27,28,29,30,31]. This approach is based on the replacement of select amino acids (AAs) in growth media with similar AAs containing non-radioactive heavy isotopes of particular atoms. These labeled (‘‘heavy’’) AAs are incorporated into newly synthesized proteins, whereas the degradation of preexisting proteins is associated with the gradual loss of proteins containing ‘‘light’’ versions of these AAs
Cells are lysed, and protein extracts are digested into short peptides, which are thereafter subjected to MS analysis
Summary
Chemical synapses contain multitudes of proteins, some of which play direct roles in synaptic transmission, whereas others regulate synaptic function or serve as structural scaffolds. Given the huge numbers of synaptic connections each central nervous system neuron makes, maintenance of synaptic contents would conceivably place enormous metabolic demands on individual neurons. These demands in turn, depend on anabolic and catabolic rates of synaptic proteins. Perhaps, the turnover kinetics of synaptic proteins have not yet been studied systematically. Whereas older studies based on radiolabeling methods indicated that the half-lives of some presynaptic proteins can be remarkably long [1,2]), more recent in vitro studies have reported half-lives of synaptic proteins in the range of several hours The metabolic cost of maintaining synapses remains largely unknown
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