Attenuated palmitoylation of serotonin receptor 5-HT1A in brain affects receptor functions and triggers depressive symptoms
The serotonergic system and in particular serotonin 1A receptor (5-HT1AR) are critically implicated in major depressive disorder (MDD), although underlying mechanisms remain enigmatic. Here we demonstrated that 5-HT1AR is palmitoylated in human and rodent brains and identified ZDHHC21 as a major palmitoyl-transferase, whose depletion reduced palmitoylation and consequently signaling functions of 5-HT1AR. Two rodent models for depression show reduced brain ZDHHC21 expression in conjunction with attenuated 5-HT1AR palmitoylation. Moreover, selective knock-down of ZDHHC21 in murine forebrain by itself sufficed to provoke depressive symptoms, demonstrating a causal relationship between 5-HT1AR palmitoylation and depression. Regarding the underlying mechanism, we identified the microRNA miR-30e as a negative regulator of Zdhhc21 expression. By analysis of the post-mortem samples from suicide MDD victims we also found ZDHHC21 expression as well as palmitoylation of 5-HT1AR to be specifically reduced within the prefrontal cortex (PFC), a brain area critically involved in the pathogenesis of depressive symptoms. Our study provides evidence for transcriptional downregulation of 5-HT1AR palmitoylation as a central mechanism in the etiology of depression and even suicide, in effect making the restoration of 5-HT1AR palmitoylation a promising clinical strategy for the treatment of major depressive disorder.
Sample Processing Protocol
Analysis of protein palmitoylation by ABE assay. To analyze palmitoylated proteins in recombinant system as well as in native tissue, the ABE assay Proteins purified after ABE approach were dissolved in 2% SDS Buffer, diluted with 19 volumes of 50mM TRIS 100mM NaCl 1mM, EDTA and digested in solution with trypsin (GOLD TRYPSIN for MS) by incubating at 37 °C for 60 min.
Data Processing Protocol
Proteins purified after from ABE approach were dissolved in 2% SDS Buffer, diluted with 19 volumes of 50mM TRIS 100mM NaCl 1mM, EDTA and digested in solution with trypsin (GOLD TRYPSIN for MS) by incubating at 37 °C for 60 min. The resulting peptide mixtures were applied to RP-18 pre-column (Waters, Milford, MA) using water containing 0.1% FA as a mobile phase and then transferred to a nano-HPLC RP-18 column (internal diameter 75 µM, Waters, Milford MA) using ACN gradient (0 – 35% ACN in 160 min) in the presence of 0.1% FA at a flow rate of 250 nl/min. The column outlet was coupled directly to the ion source of Q-exactive Orbitrap mass spectrometer (Thermo Electron Corp., San Jose, CA) working in the regime of data-dependent MS to MS/MS switch. A blank run ensuring absence of cross-contamination from previous samples preceded each analysis. All MS data sets were searched against SwissProt protein database (Swissprot 2018_02; 16,905 sequences) using the MASCOT search engine (MatrixScience, London, UK, Mascot Server 2.5.1). Both the peptide and fragment mass tolerance settings were established separately for individual LC-MS/MS runs after a measured mass recalibration, as described previously (Malinowska et al., 2012). After recalibration, the mass tolerance for proteins was in a range 5-10 ppm and for peptides 0.01-0.05 Da. The Mascot search parameters were as follows: enzyme, Trypsin; missed cleavages, 1; variable modifications N-Ethylmaleimide (C), carbamidomethyl (C), Oxidation (M); instrument, HCD; Decoy option, active. FDR was estimated with Mascot Decoy search and score threshold was adjusted for each sample to keep the FDR below 1%. Probable contaminants (e.g. keratins and albumin) were removed from the identified protein/peptide lists. For evaluation of the relative protein abundance in each sample spectral count values determined using emPAI scores were used as previously described (Sobocinska 2018 MCP, (Arike and Peli, 2014). For data sets generated by the protein-based procedure spectral counts were merged over all biological replicates. Subsequently, statistical analysis was performed on the merged data sets. For the protein data set, missing values were imputed; all zeros were replaced with 1. Only proteins which met the acceptance criteria: FDR<1%, at least two unique peptides, Mascot score over 25, non-redundant proteins, were taken for further analysis. The protein-wise natural log2of specific ratios were calculated. Significance were computed (p values) using variance t-test. Proteins with p values less than 0.05 were accepted as S-palmiotylated candidates. The statistical significance of the difference between control/scramble/shDHHC21 (enriched, reduced, no influence) was calculated using the unequal variance two-tailed heteroschedastic t-test. It was assumed that log2 shDHHC21/scramble and/or log2 shDHHC21/control and or scramble/control < -2.0 (5% threshold) and -1.0 (105 threshold) reflected reduction when >´1.0 and 2.0 reflected induction of palmitoylation of give protein. Then, statistical significance of the difference between spectral counts for each protein was estimated with the unequal variance one-tailed, heteroscedastic t-test. It was assumed that a palmitoylated protein was identified with high confidence, when the p value of the difference between two biological conditions was ≤ 0.05 or ≤ 0.001.