Comparative Phosphoproteomic Profiling of Type III Adenylyl Cyclase Knockout and Control, Male, and Female Mice

Abstract

Type III adenylyl cyclase (AC3, ADCY3 ) is predominantly enriched in neuronal primary cilia throughout the central nervous system (CNS). Genome-wide association studies in humans have associated ADCY3 with major depressive disorder and autistic spectrum disorder, both of which exhibit sexual dimorphism. To date, it is unclear how AC3 affects protein phosphorylation and signal networks in central neurons, and what causes the sexual dimorphism of autism. We employed a mass spectrometry (MS)-based phosphoproteomic approach to quantitatively profile differences in phosphorylation between inducible AC3 knockout (KO) and wild type (WT), male and female mice. In total, we identified 4,655 phosphopeptides from 1,756 proteins, among which 565 phosphopeptides from 322 proteins were repetitively detected in all samples. Over 46% phosphopeptides were identified in at least three out of eight biological replicas. Comparison of AC3 KO and WT datasets revealed that phosphopeptides with motifs matching proline-directed kinases' recognition sites had a lower abundance in the KO dataset than in WTs. We detected 14 phosphopeptides restricted to WT dataset (i.e., Rabl6, Spast and Ppp1r14a ) and 35 exclusively in KOs (i.e., Sptan1, Arhgap20, Arhgap44, and Pde1b ). Moreover, 95 phosphopeptides (out of 90 proteins) were identified only in female dataset and 26 only in males. Label-free MS spectrum quantification using Skyline further identified phosphopeptides that had higher abundance in each sample group. In total, 204 proteins had sex-biased phosphorylation and 167 of them had increased expression in females relative to males. Interestingly, among the 204 gender-biased phosphoproteins, 31% were found to be associated with autism, including Dlg1, Dlgap2, Syn1, Syngap1, Ctnna1, Ctnnd1, Ctnnd2, Pkp4, and Arvcf . Therefore, this study also provides the first phosphoproteomics evidence suggesting that gender-biased post-translational phosphorylation may be implicated in the sexual dimorphism of autism.

NMDA Receptors Control Activity Hierarchy in Neural Network: Loss of Control in Hierarchy Leads to Learning Impairments, Dissociation, and Psychosis

“…More than 90% of primary cilia in the hippocampus were ablated (see Fig. 2A) using this inducible ablation protocol, consistent with our prior report that deleted AC3 33 . For behavioral tests, mice were handled by investigators for 5 days to allow them to adjust to the investigators before starting the experiments.…”

“…Mice were maintained on a 12-h light/dark cycle at 22°C and had access to food and water ad libitum. The Ift88 floxed mouse strain [32] was cross-bred with UBC-Cre/ERT2 as previously reported (C57B1/6j genetic background, mixed sexes) [33]. At the age of 8-10 weeks, Ift88 flox/flox: UBC-Cre/ERT2 mice were orally administrated with tamoxifen (0.2 mg/g body weight, consecutive 7 days) to induce Cre recombinase expression to ablate Ift88 and generate Ift88 cilia KO mice, or with vehicle (corn oil) to make vehicle control mice.…”

Sex-Related Changes in the Clinical, Genetic, Electrophysiological, Connectivity, and Molecular Presentations of ASD: A Comparison between Human and Animal Models of ASD with Reference to Our Data

“…Several investigators have attempted to explain the male predominance of ASD by sex-specific genetic changes in single-nucleotide polymorphisms (SNPs), single-nucleotide variants (SNVs), copy-number variants (CNVs), and different sex-biased proteins [ 22 , 23 , 149 , 150 , 151 , 152 ]. Mutations in specific genes, particularly genes linked to X or Y chromosomes, have been implicated in male vulnerability to ASD [ 44 , 153 , 154 ].…”

Expression and functions of adenylyl cyclases in the CNS

“…ADCY3 mRNA is highly expressed in olfactory sensory neurons (OSN), neuronal primary cilia, and dorsal root ganglion [ 67 , 68 ]. Bulk and single-cell RNA-sequencing studies showed that ADCY3 was highly expressed in neurons, OPCs, astrocytes, pericytes and vascular fibroblast-like cells; and moderately expressed in oligodendrocytes, microglia, endothelial cells and smooth muscle cells [ 45 , 46 ].…”

“…The high expression of ADCY3 in olfactory sensory neurons suggests that it may regulate odor/pheromone detection [ 67 , 68 ]. Consistent with these results, ablation of ADCY3 leads to defective olfactory sensory neuron maturation and abnormal olfactory-based behavioral responses, including lack of preference for the test odorants in both sand-buried food task and odor-associated passive avoidance learning paradigm, absence of inter-male aggressiveness and male sexual behavior, and defective maternal behaviors [ 19 , 70 – 72 ].…”