Attenuated palmitoylation of serotonin receptor 5-HT1A affects receptor function and contributes to depression-like behaviors

Abstract

The serotonergic system and in particular serotonin 1A receptor (5-HT1AR) are implicated in major depressive disorder (MDD). Here we demonstrated that 5-HT1AR is palmitoylated in human and rodent brains, and identified ZDHHC21 as a major palmitoyl acyltransferase, whose depletion reduced palmitoylation and consequently signaling functions of 5-HT1AR. Two rodent models for depression-like behavior show reduced brain ZDHHC21 expression and attenuated 5-HT1AR palmitoylation. Moreover, selective knock-down of ZDHHC21 in the murine forebrain induced depression-like behavior. We also identified the microRNA miR-30e as a negative regulator of Zdhhc21 expression. Through analysis of the post-mortem brain samples in individuals with MDD that died by suicide we find that miR-30e expression is increased, while ZDHHC21 expression, as well as palmitoylation of 5-HT1AR, are reduced within the prefrontal cortex. Our study suggests that downregulation of 5-HT1AR palmitoylation is a mechanism involved in depression, making the restoration of 5-HT1AR palmitoylation a promising clinical strategy for the treatment of MDD.

Fig. 1

Endogenous 5-HT1AR is palmitoylated in rodent and human brains and ZDHHC21 is a major receptor palmitoyl acyltransferase. a Palmitoylation of endogenous 5-HT1AR in human, mouse, and rat brains was assessed by the acyl-biotinyl exchange (ABE) assay (see also Supplementary Fig. 1B). b Neuroblastoma N1E cells co-expressing 5-HT1AR and indicated ZDHHCs were analyzed by ABE (see also Supplementary Fig. 2A, B). c Quantification of 5-HT1AR palmitoylation after co-expression of ZDHHC5, -9, and -21 (n = 5). d N1E cells were co-transfected with hemagglutinin (HA)-tagged 5-HT1AR and indicated green fluorescent protein (GFP)-tagged ZDHHCs, followed by immunoprecipitation (IP) with anti-GFP antibody and western blot with anti-HA antibody. As a control, mixed lysates from the singly transfected cells (mix) were applied to IP. e Analysis of 5-HT1AR palmitoylation in N1E cells after knockdown of endogenous ZDHHC5, -9, and -21 by short hairpin RNA (see also Supplementary Fig. 3A–E) together with f quantification. Western blots shown in e are representative of at least four independent experiments. g Brain tissues isolated from the newborn (P0) F233Δ Zdhhc21^dep/dep (n = 7) and wild-type (n = 6) mice were collected for ABE analysis followed by quantification (see also Supplementary Fig. 3E–H). Bars show means ± SEM; *P < 0.05; **P < 0.01; one-way analysis of variance for c, f; two-tailed t test for g. Source data are available as a Source Data file

Fig. 2

Knockdown of ZDHHC5, -9, and -21 impairs 5-HT1AR-mediated signaling. a N1E cells were transfected with cAMP fluorescence resonance energy transfer-based biosensor CEPAC and 5-HT1AR-mCherry along with the indicated constructs (see also Supplementary Fig. 4A–C). After pretreatment with 1 µM forskolin and 25 µM 3-isobutyl-1-methylxanthine, cells were stimulated with 20 µM serotonin (5-HT). Each trace shows cAMP response at the single cell. b Graphs show activation time constant and c changes of cAMP response amplitude relative to pretreatment (N = 4, in each experiments at least 20 cells were analyzed; one-way analysis of variance (ANOVA)). d Analysis of extracellular signal–regulated kinase (Erk) phosphorylation in N1E cells expressing the indicated constructs after stimulation with 10 µM 5-HT. e Quantification of 5-HT evoked Erk phosphorylation calculated as the ratio of total Erk expression over the Erk phosphorylation signal. Bars show means ± SEM (N ≥ 7, one-way ANOVA; see also Supplementary Fig. 4D, E). f Representative image of hippocampal neuron expressing short hairpin RNA against ZDHHC21 together with red fluorescent protein at the day in vitro 11. Scale bar: 100 μm. g Examples of G-protein gated inwardly rectifying potassium (GIRK) channel currents in two transfected groups after application of 5-HT1AR agonist 8-OH-DPAT and channel blocker BaCl₂. h A summary of recordings from three independent culture preparations. Bars show means ± SEM of the amplitude of 8-OH-DPAT-stimulated GIRK currents. *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed t test. Source data are available as a Source Data file

Fig. 3

Palmitoylation of 5-HT1AR and expression of ZDHHC21 are decreased in the forebrains of mice with depressive-like syndrome. a Schematic diagram of experimental design for mouse model of depression. b Results of the sucrose preference test performed in control (n = 7) and chronically stressed mice (n = 19). c Immobility time in the forced swim test of control and chronically stressed mice. Group of stressed mice was then divided into anhedonic (anhed) (n = 12) and resilient (res) (n = 7) animals and immobility time was analyzed in each subgroup. One-way ANOVA. Data points represent mean ± SEM (*P < 0.05). d In anhedonic and resilient mice, palmitoylation of the 5-HT1AR was analyzed in the prefrontal cortex (PFC) and cerebellum by acyl-biotinyl exchange (see also Supplementary Fig. 5A–E). e Quantification of 5-HT1AR palmitoylation in anhedonic and resilient animals (see also Supplementary Fig. 5A–E). f Relative expression levels of ZDHHC5, -9, and -21 in the PFC of resilient and anhedonic mice assessed by quantitative reverse transcriptase-PCR. *P < 0.05; two-tailed t test. (See also Supplementary Fig. 5F–H). Source data are available as a Source Data file

Fig. 4

Palmitoylation of 5-HT1AR and expression of ZDHHC21 are decreased in the forebrains of rats with depression-like phenotype. a Schematic diagram of experimental design for restraint stress depression model in Wistar rats. b Normalized corticosterone blood level of the control (ctrl) and rats with depression-like behavior (depr). c Results of the sucrose preference test in control and chronically stressed rats. d The duration of immobility in the forced swim test of the control and rats with depression-like behavior. e Body weight gain during experiment of the control and rats with depression-like behavior. Data points in b–e represent mean ± SEM. (n = 10 for each group; *P < 0.05; **P < 0.01; ***P < 0.001). f Prefrontal cortex (PFC) and cerebellum of the control Wistar rats (ctrl) and rats with depression-like behavior (depr) were subjected to acyl-biotinyl exchange to visualize 5-HT1AR palmitoylation. g Bar graphs representing normalized level of 5-HT1AR palmitoylation in control (n = 5) and rats with depression-like behavior (n = 5) animals. All blots are representative of at least three independent experiments. Data points represent the means ± SEM. *P < 0.05; ***P < 0.001, two-tailed t test (see also Supplementary Fig. 6A–E). h Relative expression levels of ZDHHC5, -9, and -21 in the PFC of control rats and rats with depression-like phenotype (two-tailed t test, see also Supplementary Fig. 6F, G). Source data are available as a Source Data file

Fig. 5

Selective knockdown of ZDHHC21 in the prefrontal cortex (PFC) leads to development of depressive-like behavior in mice. a Cortical slice of the C57BL/6J mouse 30 days after administration of adenovirus-associated virus encoding for short hairpin RNA (shRNA) against ZDHHC21 along with red fluorescent protein (see also Supplementary Fig. 7A, B, E). Scale bar: 200 μm. b Relative expression levels of Zdhhc21 in the PFC after injection of the indicated constructs (n = 4 for each group, one-way analysis of variance (ANOVA)). c PFCs of mice were isolated 30 days after injection either with vehicle or scrambled shRNA or shRNA against ZDHHC21 and subjected to acyl-biotinyl exchange assay to define 5-HT1AR palmitoylation. Quantification of palmitoylation (d) and expression (e) of 5-HT1AR in the PFCs of mice after injection either with vehicle or scrambled shRNA or shRNA against ZDHHC21. f Immobility time in the forced swim test of mice treated with vehicle (n = 10), shRNA against ZDHHC21 (n = 9), or scrambled shRNA (n = 11). Statistical significance between values is noted (*P < 0.05, **P < 0.01; one-way ANOVA; see also Supplementary Fig. 7J–M). g Comparison of the motor activity assessed by the open field test in mice treated with vehicle, shRNA against ZDHHC21, or scrambled shRNA. h Volcano plot depicting differential S-palmitoylation analysis between mice injected with scramble shRNA and with shRNA against ZDHHC21 using mass spectrometry spectral counts data with estimated fold changes (x axis) versus the significance, i.e., −log₁₀ (P values) for each protein (y axis). Dotted vertical lines lineate events with fold change >2. Dotted horizontal lines denote point at which two-tailed t test P > 0.05. Value for 5-HT1AR is marked in red (n = 3 biological replicates for each condition; see also Supplementary Data 1 and 2). 1, TRPC channel subfamily V (TRPV2); 2, phosphoglycerate mutase 1 (PGAM1); 3, proteosomal ubiquitin receptor ADRM1; 4, exopolyphosphatase PRUNE1; 5, signal transducer and activator of transcription 6 (STAT6); 6, Hsc70-interacting protein. Source data are available as a Source Data file

Fig. 6

Attenuated 5-HT1AR palmitoylation in the prefrontal cortex (PFC) of individuals with major depressive disorder (MDD) who died by suicide correlates with reduced expression of ZDHHC21. a 5-HT1AR palmitoylation in the PFC and cerebellum from the control and individuals with MDD who died by suicide (PFC, n = 5; cerebellum, n = 4). b Normalized level of 5-HT1AR palmitoylation in the PFC and cerebellum of individuals with MDD who died by suicide in comparison with control subjects (two-tailed t test; see also Supplementary Fig. 8). c Relative expression levels of ZDHHC5, -9, and -21 in the PFC (left) and cerebellum (right) of control (n ≥ 14) and individuals with MDD who died by suicide (n ≥ 12). Data points represent the means ± SEM (*P < 0.05; ***P < 0.001; two-tailed t test). d N1E cells were transfected with microRNAs as indicated and the expression of ZDHHC21 was determined by reverse transcriptase-PCR (see also Supplementary Table 1). Data points represent mean ± SEM from at least three independent experiments (*P < 0.05; ***P < 0.001; two-tailed t test). e N1E cells were transfected with 5-HT1AR along with miR-30e, miR30-a, or shRNA against ZDHHC21. Palmitoylation of 5-HT1AR was analyzed by acyl-biotinyl exchange. Western blots shown are representative of at least three independent experiments. f Analysis of miR-30a, -30e, and -200a expression in the PFC of control and individuals with MDD who died by suicide. (*P < 0.05, **P < 0.01; two-tailed t test). Source data are available as a Source Data file