Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover

Abstract

Memory formation is believed to result from changes in synapse strength and structure. While memories may persist for the lifetime of an organism, the proteins and lipids that make up synapses undergo constant turnover with lifetimes from minutes to days. The molecular basis for memory maintenance may rely on a subset of long-lived proteins (LLPs). While it is known that LLPs exist, whether such proteins are present at synapses is unknown. We performed an unbiased screen using metabolic pulse-chase labeling in vivo in mice and in vitro in cultured neurons combined with quantitative proteomics. We identified synaptic LLPs with half-lives of several months or longer. Proteins in synaptic fractions generally exhibited longer lifetimes than proteins in cytosolic fractions. Protein turnover was sensitive to pharmacological manipulations of activity in neuronal cultures or in mice exposed to an enriched environment. We show that synapses contain LLPs that may underlie stabile long-lasting changes in synaptic structure and function.

Keywords: enriched environment; long-lived proteins; mass spectrometry; neuronal activity; protein turnover.

Fig. 1.

Metabolic labeling in mice and identification of synaptic LLPs. (A, Left) Schematic of metabolic labeling in mice. At 3 wk old, male mice were fed with chow containing heavy-isotope-labeled Lys-¹³C₆ for 7 wk, followed by unlabeled chow for 7 wk. Mice were exposed to either control or EE for 3 d before chasing with unlabeled chow. Mice were killed at the end of the pulse or chase periods followed by tissue isolation, subcellular fractionation, and analysis by LC-MS/MS. (A, Right) Stable proteins incorporate less label during pulse but retain more during chase. Unstable proteins rapidly acquire and then lose heavy label. (B) RIA of heavy lysine incorporated in the proteome during the pulse of forebrain synaptosomes (Syn.), cytosol, and or whole kidney. Box plot shows high levels of heavy labeling were achieved on average; individual protein species show variable labeling according to their turnover. Labeling efficiency was significantly higher in the kidney compared with brain, indicating higher rates of protein metabolism (P < 0.05, Student’s t test). Data obtained from eight mice. Error bars indicate the full distribution of data points. (C) Subcellular fractionation of mouse forebrain was monitored by Western blot analyses of synaptic and nonsynaptic proteins. Whole forebrain lysate, cytosol, and synaptosomal fractions (Syn.) were analyzed.

Fig. 2.

Characterization of synaptic LLPs. (A) Examples of decline in heavy label during chase, normalized to pulse. The decrease in heavy label-containing peptides during the chase period is used to indicate protein turnover, with greater decline indicating higher turnover. Data obtained from four mice from control group. (B) Plot indicates ranked turnover ratio for 2,272 proteins. The turnover ratio is calculated as the RIA_pulse/RIA_chase. A value close to 1 indicates slow turnover, and larger values indicate greater protein turnover. (Inset) The 164 proteins defined as LLPs with a turnover ratio of 2 or less. Data obtained from four mice from control group. (C) MS1 extracted-ion chromatogram of representative peptides of LLPs from the chase period. Intensities of isotopes from eluted peptides were aligned according to their retention time and plotted for representative LLP peptides for CRMP5, CRMP3, Tubb2a, RRas2, Prkar2b, and Sh3gl3. Light isotope signal is plotted in black and heavy signal in red. (D) Chart indicates molecular functions and cellular components of 164 synaptosomal LLPs defined by at least 50% heavy label accumulated during pulse remaining after chase.

Fig. 3.

Synaptosome proteins are stabilized relative to cytosolic proteins. (A) Western blot of forebrain subcellular fractions. Many LLPs are localized in both cytosol and synaptosomal fractions (Syn.). (B) Plot indicates the ranked protein turnover ratios of synaptosome and cytosol proteomes plotted against the fraction of the total number of proteins identified. Cytosolic proteins showed a statistically significant increase in protein turnover under control conditions (P < 0.001, Student’s t test). (C) Plot indicates the ranked protein turnover from synaptosomal fraction and the paired values of proteins from cytosolic fraction. The spread indicates that turnover ratio of certain proteins was disproportionally affected by their subcellular localization. Data obtained from four control mice.

Fig. 4.

Protein turnover is accelerated by EE experience. (A) Plot indicates the ranked turnover ratios of synaptosomal and cytosolic proteins from control mice and mice that underwent 3 d of EE exposure. EE exposure resulted in an almost uniform increase in protein turnover that was statistically significant (P < 0.001, Student’s t test). Data obtained from four control mice and four EE mice. (B) Plot indicates the ranked protein turnover from control mice and the paired values obtained from EE mice. The spread indicates that certain proteins from synaptosomal and cytosolic fractions were disproportionally affected by EE exposure. Data obtained from four control mice and four EE mice.

Fig. 5.

Protein turnover in cultured neurons is regulated by neuronal activity. (A) Schematic of metabolic labeling in cultured neurons. Rat cortical neurons were grown for 11 d in vitro (DIV11) under normal conditions. The media was then spiked with a 5× excess of heavy isotope labeled lysine and arginine with or without the addition of Bic (20 μM) or TTX (1 μM). Whole-cell lysate was obtained at indicated times following treatment and analyzed by MS. (B) Plot indicates the ranked RIA for 2,593 proteins identified/quantified in all four time points as indicated. RIA increases steadily with time after labeling, approaching the maximum RIA value of 0.833. Proteins with slower turnover incorporate less isotope label while proteins with higher turnover rapidly acquire the isotope label. The rate of isotope incorporation is used to calculate half-life. (C) Plot indicates ranked half-lives in days for 2,593 proteins from control-treated neurons. Half-lives calculated from four separate time points. (D) Plot indicates the molecular functions and cellular components for 117 proteins with a half-life of 5 d or longer. (E) Plot indicates ranked half-lives for 2,245 proteins from control-, Bic-, and TTX-treated neurons. Half-lives calculated from four separate time points. TTX resulted in a statistically significant shift toward longer protein half-lives (P < 0.001, Student’s t test), while Bic treatment caused a slight decrease in overall protein half-lives that was not significant (Student’s t test). (F) Plot indicates the fold change in protein half-life from Bic-treated neurons over control ranked from proteins stabilized by Bic (Bic/con > 1) to those destabilized (Bic/con < 1). The dashed line indicates the control protein half-life. (G) Plot indicates the fold change in protein half-life from TTX-treated neurons over control ranked from proteins stabilized by TTX (TTX/con > 1) to those destabilized (TTX/con < 1). The dashed line indicates the control protein half-life.