Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress

Abstract

Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.

Figure 1. Otp Mediates Stress Response

(A) Coronal sections (6 μm) through the PO showing colocalization of Otp and crh in a 6-day-old zebrafish larvae. Larvae were subjected to whole-mount in situ hybridization with a crh-directed probe followed by sectioning and immunostaining with an anti-Otp antibody. High magnification of the crh⁺;Otp⁺ area (black rectangle) is shown in the inset. (B) Histogram showing average cell counts in individual CRH⁺ neuronal clusters of a null zebrafish mutant allele, otpa^m866−/− (n = 20) compared to heterozygous otpa^m866−/+ siblings (n = 19). ns, not significant. (C–F) Projected confocal z stack images of crh neurons in otpa^m866−/− (D and F) and otpa^m866−/+ siblings (C and E), acquired from lateral (C and D) and dorsal (E and F) angles. Neurons were visualized by fluorescent in situ hybridization of crh mRNA. (G) Induction of crh mRNA by homeostatic challenge. Physical stressor was applied to 6-day-old progenies of an otpa^m866−/+ cross for a period of 4 min. The amount of crh mRNA was measured in individual fish larvae at different time points of recovery using quantitative PCR (qPCR). Each tested larva was then genotyped by sequencing and crh mRNA levels of mutant (^−/−) and heterozygous (^−/+) animals were plotted accordingly. *p < 0.05; n = 8. (H) Gain of function of Otpa in Otp-positive neurons. Physical stress challenge was applied to either a transgenic otp:Gal4 embryos expressing Gal4 in Otp⁺ cells or their siblings, which were injected with a transposonbased plasmid vector containing the full-length cDNA encoding the otpa gene under the control of ten UAS elements (otp:Gal4 >> UAS:otpa). The amount of crh mRNA was measured using qPCR. *p < 0.05, n = 8. The following abbreviations are used: e, eye; HB, hindbrain; PO, preoptic area; PT, posterior tuberculum; RQ, relative quantity; Tel, telencephalon. Scale bar represents 50 μm in (A) and 100 μm in (C)–(F).

Figure 2. Otp Is Necessary for Novelty Stress Response

(A) The “novel tank” test measures the vertical place preference of adult zebrafish following exposure to a novel environment. Four-month-old otpa^m866−/− and their WT siblings were moved from their home tank into a new tank with dissimilar dimensions, and their behavior was monitored for a period of 6 min using an automated video tracking system. (B and C) Quantified behavioral parameters showing locomotor activity (B) and the time spent in the top and bottom thirds of the test tank (C) within consecutive time intervals (2 min, each). *p < 0.05; n = 11.

Figure 3. ChIP Assay Reveals Otp Association with Stress Promoters

(A) Schematic representation of the experimental setup used for the ChIP-seq screen. A pool of fish (600 larvae per lane) were subjected to stress challenges in bulk or left unchallenged followed by anti-Otp ChIP. Putative Otp targets induced by stress were identified either by high-throughput sequencing or by qPCR with promoter-specific primers. (B and D) Histograms showing quantitative ChIP analyses of the recruitment Otp to crh (*p < 0.05, n = 3) and a2bp1 (*p % 0.1, n = 3) promoters. Chromatin was extracted from a pool of 50 larvae per treatment 30 min after physical or osmotic challenges, followed by anti-Otp ChIP. Recruitment of Otp to the respective promoter was calculated relative to the amount of input chromatin. (C) Stressor-induced Otp binding peaks (arrowheads), which were mapped at the vicinities of a2bp1 gene loci. Bar histograms represent the abundances of high-throughput sequencing reads in relation to their chromosomal location.

Figure 4. Splicing Factor A2BP1/Fox-1 Is Induced by Stressors

(A and B) Acute stress paradigm performed on mice. A computer-controlled system was used to deliver a foot shock stress challenge to mice placed in a chamber. Mice were taken out of the chamber, PVN punches were dissected from mice at different time points following stress initiation, and crh (A) or a2bp1 (B) mRNA levels were analyzed by qPCR. *p < 0.05, n = 5. RQ, relative quantity. (C) Physical stress challenges were applied, in bulk, to 6-day-old zebrafish larvae derived from an otpa^m866−/+ cross. The amount of a2bp1 mRNA was measured in individual fish larva at different time points of recovery using qPCR. Each tested larva was then genotyped by sequencing and a2bp1 mRNA levels of mutant (^−/−) and heterozygous (^−/+) animals were plotted accordingly. *p < 0.05; n = 4.

Figure 5. pCREB-Otp Protein Complex Is Recruited to crh and a2bp1 Promoters

(A) A pool of 100 larvae per lane were subjected to stress challenges in bulk or left unchallenged. Larvae were lysed and subjected to IP with a resin-coupled anti-Otp antibody (Ab) followed by gel electrophoresis and protein electrotransfer to a membrane. Nitrocellulose filters were immunoblotted with an antibody directed to pCREB, stripped, and reblotted with an anti-Otp antibody. (B) Schematic representation of the sequential ChIP- reChIP procedure in which larvae lysates were first subjected to anti-Otp ChIP and the resulting Otp-DNA complex were eluted and subjected to a second ChIP with a pCREB antibody. (C) A pool of fish (300 larvae per lane) were subjected to stress challenges in bulk or left unchallenged followed by the ChIP-reChIP procedure using control IgG, anti-Otp, and anti-pCREB antibodies. The histograms show promoter co-occupancy analysis of Otp and pCREB (n = 2).

Figure 6. Modulation of Stress Response by Alternative Splicing of PAC1

(A) Quantitative PCR analysis of the two pac1 splice isoforms in mice that were subjected to a foot shock challenge. PVN punches were harvested at different time points following stress initiation. The analyzed pac1 cDNA fragments corresponding to the short and long (hop) splice isoform are schematically shown. The hop cassette is encoded by exon 14 (E14). *p < 0.05, n = 11. (B) Calculated ratio between the amounts of the long and short pac1 isoforms shown in (A) as a function of time after the initiation of the foot shock stressor showing a positive linear correlation. R² = 0.9, n = 11. (C) The top part shows a scheme depicting pac1a gene structure including the respective binding sites for two antisense MO oligonucleotides designed either to block expression of both PAC1 isoforms (pac1a-ATG MO) or prevent the inclusion of the hop exon by alternative splicing (pac1a-hop MO). The bottom part shows a RT analysis of 6-day-old larvae showing the effect of the pac1a-hop MO. The long (hop) and short splice variants (arrows) are visualized in the control larvae, whereas only the short isoform is present in pac1a-hop MO-injected animals. Inclusion of adjacent constitutive exons is not affected by the pac1a-hop MO. (D–F) Quantitative PCR analyses of crh mRNA following a physical stress challenge of 6-day-old larva. The amount of crh mRNA was measured in individual fish larvae at different time points of recovery. (D) Stress challenge was applied to either mock-treated 6-day-old larvae (control, n = 14) or their siblings that were injected with antisense pac1a-ATG MO (n = 12), which prevents the formation of both PAC1 isoforms. Alternatively, transgenic otp:Gal4 embryos expressing Gal4 in Otp⁺ cells were coinjected with pac1a-ATG MO together with transposon-based vector constructs harboring PAC1’s short (pac1a-ATG MO+PAC1-short, n = 4) and long (pac1a-ATG MO+PAC1-long, n = 4) isoforms under the control of ten UAS elements (UAS:PAC1-short or UAS:PAC1-long, respectively). *p % 0.01. (E) Stress challenge was applied to either mock-treated 6-day-old transgenic otp:Gal4 larvae (control, n = 11) or their siblings, which were injected with a pac1ahop MO (n = 12) or pac1a-ATG MO together with UAS:PAC1-long construct (pac1a-ATG MO+PAC1-long, n = 6). *p % 0.05. (F) Stress challenge was applied to either a otp:Gal4 transgenic line larvae (control, n = 10) or their siblings, which were injected with constructs containing the short (n = 7) or long (n = 6) PAC1 isoform under the control of ten UAS elements (otp:Gal4 >> UAS:PAC1-short or otp:Gal4 >> UAS:PAC1-long, respectively). The amount of crh mRNA was measured as described above. *p < 0.05. (G) Analysis of cortisol content following a physical stress challenge applied to either mock-treated 6-day-old larvae (control) or their siblings, which were injected with pac1a-hop MO. Whole-body cortisol levels were measured at different time points of recovery in tissue extracts derived from pools of ten larvae. *p < 0.05, n = 3. The following abbreviations are used: AS, alternative spliced exon; MO, morpholino; ns, not significant; RQ, relative quantity.

Figure 7. Anxiety-like Behavior following Inhibition of PAC1 Splicing

(A) Schematic representation of the two-compartment arena used for the “light-dark preference” behavioral test of 6-day-old larvae (adopted from [Steenbergen et al., 2011]). The assay measures anxiety-like dark avoidance behavior. (B) Histogram showing the percentage of time spent on the black side of the two-compartment light-dark measuring arena. Treatment of larvae with 5 μm Diazepam decreases dark avoidance in a light-dark box. *p < 0.05, n = 24. (C and D) Control (n = 36) and pac1a-hop morphant (n = 33) larvae were challenged with an osmotic stressor for 4 min, and their total distance swum in the test arena (C) and dark avoidance (D) were measured for 5 min at 0, 5, 60, and 120 min time points of recovery from osmotic shock. **p < 0.01; ns, not significant. (E) A model summarizing the roles of Otp and alternative splicing in stress adaptation. The stereotypic kinetics of stress-induced crh mRNA levels is shown on the left, whereas the genetic and biochemical interactions are illustrated on the right. Otp and the short PAC1 isoform (a receptor for the PACAP neuropeptide) promote crh transcription, whereas the generation of the long PAC1 (hop) alternative (alter.) splice variant terminates crh transcription at the late recovery phase (see text).