Western and Mediterranean diets differentially affect cerebral cortical gene expression, brain structure, and socioemotional behavior in middle-aged, female nonhuman primates (Macaca fascicularis). Here we quantified the impact of these diets on the presynaptic proteome of approximately 3.6 million synapses isolated from the lateral temporal cortex with synaptometry time of flight (SynTOF) mass spectrometry, a powerful technology for quantifying the molecular diversity of single presynapses. Associations between the presynaptic proteome, adjacent cortex transcriptome, and multi-system phenotypes were assessed using a machine learning approach. Subjects were socially housed macaques (Macaca fascicularis) randomized to eat Mediterranean (n=17) or Western (n=19) diets for 31 months prior to brain retrieval. Six presynaptic proteins (DAT, Aβ42, calreticulin, LC3B, K48-Ubiquitin, SLC6A8) were elevated in the presynaptic proteome by the Mediterranean compared to the Western diet (p<0.05). Transcriptomic data from adjacent cortex predicted all the SynTOF markers. The SPATA22 transcript was positively correlated with three SynTOF markers (LRRK2, TMEM230 and Aβ40) (all p<0.05), while TFAP2C was positively correlated with SynTOF markers pTau, CD47, PARKIN and GAD65 (p<0.05). The multi-system phenotypes significantly predicted 26 SynTOF markers. The strongest relationships were between synaptosome GFAP and MRI-determined changes in white matter (p<0.001). Numerous SynTOF markers were inversely correlated with hepatosteatosis (e.g., Aβ42, Aβ40, DAT and K48-Ubiquitin adjusted p’s<0.05), suggesting relationships between liver health and the presynapse proteome. SynTOF markers were also associated with behavioral and physiological measures of social environmental stress. Together these observations demonstrate that diet composition drives temporal presynaptic protein composition, that transcriptional profiles strongly predict the presynaptic proteomic profile, and that presynaptic proteins were closely associated with peripheral metabolism, stress responsivity, neuroanatomy, and socioemotional behavior. These data demonstrate brain phenotypes and brain-body interactions which can be influenced by diet composition, with potential implications for nutritional approaches to brain and body health.

Synaptometry by time-of-flight (SynTOF) mass spectrometry. Following our established and published protocol for a SynTOF panel^1,2, synaptosome samples were prepared, barcoded, and stained. Mass Synaptometry data were acquired, debarcoded, and sequentially gated for % negative and positive events. positive events were selected by CD56 and SNAP25, and nonpresynaptic events selected by CD11b, gephyrin, PSD95, and MBP. Nonpresynaptic events were removed from downstream analyses. Synaptosome processing and analysis pipelines were previously described by us at ^2,3,4.

[1] Gajera CR, Fernandez R, Postupna N, et al. Mass synaptometry: High-dimensional multi parametric assay for single synapses. J Neurosci Methods 2019;312:73-83.

[2] Gajera CR, Fernandez R, Montine KS, et al. Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events. Cytometry A 2021;99:939-945

[3] Berson E, Gajera CR, Phongpreecha T, et al. Cross-species comparative analysis of single presynapses. Sci Rep 2023;13:13849.

[4] Gajera CR, Fernandez R, Postupna N, et al. Mass Synaptometry: Applying Mass Cytometry to Single Synapse Analysis. Methods Mol Biol 2022;2417:69-88.