PAC1 Receptor Knockout Mice Reveal Critical Links Between ER Stress, Myelin Homeostasis, and Neurodegeneration

Abstract

The pituitary adenylate cyclase-activating polypeptide receptor 1 (PAC1) plays a pivotal role in central nervous system development and homeostasis. Comparisons of PAC1 knockout (PAC1^-/-), heterozygous (PAC1^+/-) and wild-type (PAC1^+/+) mice demonstrate that PAC1 deficiency severely impairs pre-weaning survival and results in marked developmental deficits, including reduced postnatal weight and altered locomotor behavior. PAC1^-/- mice exhibited hyperlocomotion, reduced anxiety-like behavior, and transient deficits in motor coordination. Gene expression analyses revealed widespread dysregulation of oligodendrocyte-associated markers, with significant myelin reduction and decreased mature oligodendrocyte density in the corpus callosum. ER stress was evidenced in both white matter and motor cortex, as indicated by altered expression of UPR-related genes and increased phosphorylated (p)IRE1⁺ neurons. Retinal morphology was compromised in PAC1^-/- animals, with reduced overall retinal and ganglion cell layer thickness. Notably, no gross morphological or molecular abnormalities were detected in the spinal cord regarding myelin content or MBP expression; however, synaptic marker expression was selectively reduced in the ventral horn of PAC1-deficient mice. Together, these findings highlight a critical role for PAC1 in oligodendrocyte maturation, retinal development, and synaptogenesis, providing new insights with relevance in multiple sclerosis and other neurodevelopmental and neurodegenerative conditions.

Keywords: PAC1 receptor; endoplasmic reticulum (ER) stress 2; multiple sclerosis; myelin homeostasis; neurodegeneration; oligodendrocytes; unfolded protein response (UPR).

Figure 1

Preweaning deaths and post-weaning survival rates in PAC1^+/+ (top panel), PAC1^+/− (middle panel) and PAC1^−/− mice (bottom panel). Data shown in each pie chart indicates the percentage of mice that succumbed preweaning over the total mice bred for that specific genotype. Total number of mice born per each genotype from January until December 2024 was: PAC1^+/+ mice = 83 (preweaning deaths = 10), PAC1^+/− mice = 74 (preweaning deaths = 9), and PAC1^−/− mice = 80 (preweaning deaths = 31).

Figure 2

Effects of partial and complete PAC1 deletion on body weight, balance, coordination, and locomotion. (A) Average daily weights ± SEM of PAC1^+/+, PAC1^+/− and PAC1^−/− mice during the testing period (4 weeks). **** p < 0.0001 vs. PAC1^+/+ or PAC1^+/− mice, as computed using repeated measures ANOVA and Tukey post hoc analysis. (B) Schematic of the experimental timeline, starting at birth of the pups, followed by the onset of behavioural assessments at 8 weeks of age, and concluding with tissue collection at 12 weeks. (C) Latency to fall of mice undergoing weekly Rotarod tests. Results shown are the mean latency to fall (in seconds) ± SEM of PAC1^+/+ (n = 6), PAC1^+/− (n = 8) and PAC1^−/− (n = 6) mice. (D) Representative line track and (E) total distance travelled by mice of each genotype in the Open Field arena each week (over a 3 min testing period), and (F) time spent in centre. * p < 0.05 or ** p < 0.01 vs. PAC1^+/+ mice, as determined using a linear mixed-effects model using genotype and time as fixed effects and mouse identification as a random effect. Post hoc comparisons were corrected using Tukey method. ns = not significant.

Figure 3

Effects of global PAC1 deficiency on the expression of myelin-associated genes, myelin and mature oligodendrocyte density. Analyses of PLP (A,E), MBP (B,F), MOG (C,G) and Olig2 (D,H) gene expression in the CNS white matter and motor cortex, respectively, of PAC1^+/+, PAC1^+/− and PAC1^−/− mice. Results are presented as the mean fold change ± SEM from n = 6–8 mice per genotype. Representative Luxol Fast Blue (LFB) staining (with insets at higher magnification), showing myelin density in the corpus callosum (I) and hippocampus/cortex (J) of mutant mice. Quantification of the mean myelin intensity ± SEM (n = 6 mice per group) in the corpus callosum of mice, performed via LFB staining (K). Representative immunofluorescence staining of ASPA+ cells (with insets at higher magnification) of the corpus callosum of wild-type (PAC1^+/+), heterozygous (PAC1^+/−) and homozygous PAC1 knockout mice (PAC1^−/−) (L). Stereology of the average ASPA⁺ cell density (cells/mm²) ± SEM (n = 3–7 mice per group) (M). * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001 vs. PAC1^−/− (or PAC1^+/−, as indicated) after analysis using One-Way ANOVA and Tukey’s multiple comparisons tests. ns = not significant.

Figure 4

Effects of partial and complete PAC1 gene deletion on UPR activation. Gene expression of (A,E) PERK, (B,F) ATF4, (C,G) DDIT3 and (D,H) ATF6 in the white matter and motor cortex of PAC1^+/+, PAC1^+/− and PAC1^−/− mice. Results presented are the mean fold change ± SEM (n = 6 mice per genotype). (I) Representative photomicrographs (with insets at higher magnification taken from the dashed white areas) of the motor cortex (approximate boundaries of cortical layers I–VI are indicated) that have been co-stained with pIRE1 and TUJ1 and counterstained with the nuclear dye DAPI and (J) related stereological measures of the ratio of pIRE1⁺ neurons (TUJ1⁺ cells) in each region of interest (each ROI = 40,000 µm², n = 6 mice). Data shown is the average percentage of pIRE1+/TUJ1+ cells per ROI ± SEM from 6 mice per group. (K) Real-time qPCR analysis of ERN1 gene expression in the motor cortex of mutant mice. Each data point is the mean ± SEM (n = 6 mice). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. PAC1^+/+ (or PAC1^+/−, as shown), as determined using One-Way ANOVA followed by Tukey’s multiple comparisons test. ns = not significant.

Figure 5

Gross morphology and morphometrical examination of the retina in PAC1^−/− and PAC1^+/− mice. Representative brain sagittal sections of PAC1^+/+, PAC1^+/− and PAC1^−/− mice (A) and retinal cross sections depicting the different layers after staining using H&E (B). Individual layers are labelled as follows (from top to bottom): Chor = choroid, RPE = Retinal pigmented epithelium OS/IS = Outer/Inner segment of the Rode and Cone layer, ONL = Outer Nuclear Layer, OPL = Outer Plexiform Layer, INL = Inner Nuclear Layer, IPL = Inner Plexiform Layer, GCL = Ganglionic Cell Layer. Violin plots showing the average retinal thickness ± SEM (n = 5 mice per group) (C) and that of the GCL (n = 5) (D). * p < 0.05 or *** p < 0.001 vs. PAC1^−/− or PAC1^+/− mice, as calculated using One-Way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 50 µm.

Figure 6

Myelin density and MBP expression in the spinal cord of PAC1^−/− mice. Representative LFB-stained cross sections of the spinal cord of PAC1^+/+, PAC1^+/− and PAC1^−/− mice (A) and related quantifications of mean LFB density ± SEM (9–10 sections per group from n = 4–5 mice) (B). Real-time qPCR analyses of MBP gene expression in mutant mice. Results are the average fold change ± SEM (n = 6 mice per group) (C,D). Representative Western blot of MBP expression (E) and related densitometry across the different genotypes (n = 6 mice per group). Data shown is the mean ± SEM (F). ns = not significant. GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

Expression of synapse-associated markers in the spinal cord of PAC1^+/+, PAC1^+/− and PAC1^−/− mice. Representative Synapsin II immunoreactivity (IR) in spinal cord cross-sections from PAC1^+/+, PAC1^+/− and PAC1^−/− mice (A) and related quantifications of the mean Synapsin II IR ± SEM (6–10 sections from n = 4–5 mice per genotype) from the dorsal (B) and ventral horns (C). Western blot showing Synaptophysin protein expression (D) and Synapsin II (showing both IIa and IIb isoforms) (E) and corresponding densitometric analyses using spinal cord lysates from mutant mice (n = 6 mice per genotype). Results shown in bar graphs are the mean ± SEM (D,E). * p < 0.05, ** p < 0.01 vs. PAC1^+/+ mice, as determined by One-way ANOVA and Tukey post hoc test. ns = not significant.