Gender-different effect of Src family kinases antagonism on photophobia and trigeminal ganglion activity

Abstract

Background: Src family kinases (SFKs) contribute to migraine pathogenesis, yet its role in regulating photophobia behaviour, one of the most common forms of migraine, remains unknown. Here, we addressed whether SFKs antagonism alleviates photophobia behavior and explored the underlying mechanism involving hypothalamus and trigeminal ganglion activity, as measured by the alteration of neuropeptide levels and transcriptome respectively.

Methods: A rapid-onset and injury-free mouse model of photophobia was developed following intranasal injection of the TRPA1 activator, umbellulone. The role of SFKs antagonism on light aversion was assessed by the total time the mouse stays in the light and transition times between the dark and light compartments. To gain insight to the preventive mechanism of SFKs antagonism, hypothalamic neuropeptides levels were assessed using enzyme linked immunofluorescent assay and trigeminal ganglion activity were assessed using RNA-sequencing and qPCR analysis.

Results: SFKs antagonism by a clinically relevant SFKs inhibitor saracatinib reduced the total time in light and transition times in male mice, but not in females, suggesting SFKs play a crucial role in photophobia progressing and exhibit a male-only effect. SFKs antagonism had no effect on hypothalamic calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide levels of all mice investigated, suggesting the gender-different effect of saracatinib on light aversion appears to be independent of these hypothalamic neuropeptide levels. In trigeminal ganglion of male mice, photophobia is associated with profound alteration of differentially expressed genes, part of which were reversed by SFKs antagonism. Subsequent qPCR analysis showed SFKs antagonism displayed gender-different modulation of expression in some candidate genes, particularly noteworthy those encoding ion channels (trpm3, Scn8a), ATPase signaling (crebbp, Atp5α1) and kinase receptors (Zmynd8, Akt1).

Conclusions: In conclusion, our data revealed that SFKs antagonism reduced photophobia processing in male mice and exhibited gender-different modulation of trigeminal ganglion activity, primarily manifesting as alterations in the transcriptome profile. These findings underscore the potential of SFKs antagonism for allieving photophobia in males, highlighting its value in the emerging field of precision medicine.

Keywords: Gender; Light aversion; Migraine; Src family kinases; Transient receptor potential ankyrin 1; Trigeminal ganglion.

Fig. 1

Experimental flowchart showing light aversion behavior induction paradigm. On day 1, each mouse was acclimated in the testing chamber with the light (400–500 lx) on for 35 min twice and i.p. administration of the test drugs was conducted in between the acclimation. On day 2, each mouse was acclimated with the same timeline as on day 1: Mouse behavior was recorded in the testing chamber for 35 min under light intensity of 27,000 lx [17] as the self-control. The mouse was then i.p. administered with the test drugs, immediately followed by i.n. administration of either 1 µmol/kg UMB or its vehicle 0.2% dimethyl sulfoxide (DMSO). Subsequently, mouse light aversion behavior was recorded for another 35 min. After light aversion recording, mice were immediately sacrificed by rapid cervical dislocation, dissected for the hypothalamus and TG. Due to the small amounts of tissue, the left and right hypothalamus and TG of each mouse were merged respectively, rapidly homogenated within 15 s in liquid nitrogen, aliquoted and stored in -80 degree for subsequent analysis

Fig. 2

SFKs antagonism by SRCT displayed gender different effects on the total time in light in C57BL/6J photophobia mice induced by intranasal injection of UMB. Light aversion was induced by intranasal injection (i.n.) of UMB (150 µg/kg). 0.2% DMSO was injected as the control. There was total three experimental groups in each sex: (1) Control (0.25% DMSO, vehicle, i.p.), photophobia (0.25% DMSO, vehicle, i.p.) and photophobia with the SFKs inhibitor, SRCT (20 mg/kg, i.p.) groups. SRCT or its vehicle was intraperitoneally injected for 2 consecutive days prior to photophobia induction. Light aversion behavior of each mouse was recorded before and after light aversion induction for self-comparison in both male (M) and female (F) mice. (A, B) Total time in light (sec) before and after light aversion induction. (C) Total time in light recorded after light aversion induction relative to self-control. (D) Comparison of total time in light (sec) recorded after light aversion induction between male and female mice. (E) Comparison of total time in light relative to self-control recorded after light aversion induction between male and female mice. Data are shown as mean ± SEM. Significant difference was shown by * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001. * Indicates comparison between dependent groups and ^# indicates comparison between independent groups

Fig. 3

SFKs antagonism by saracatinib reduced transition times between the light and dark compartments in light in male C57BL/6J photophobia mice induced by intranasal injection of UMB. Transition times data was collected as the same as total time in light from the same mice shown in Fig. 2. (A, B) Transition (times) between the light and dark compartments before and after light aversion induction. (C) Transition times recorded after light aversion induction relative to self-control. (D) Comparison of transition times recorded after light aversion induction between male (M) and female (F) mice. (E) Comparison of transition times recorded after light aversion induction relative to self-control between male and female mice. Data are shown as mean ± SEM. Significant difference was shown by * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001; ^#P < 0.05, ^##P < 0.01. *Indicates comparison between dependent groups. ^# Indicates comparison between independent groups

Fig. 4

SFKs antagonism did not alter CGRP and PACAP levels in the hypothalamus of C57BL/6J photophobia mice. The hypothalamus of the mouse in the control, photophobia, and photophobia with SRCT groups in both male and female mice was collected from the same mouse cohort as the light aversion behavior shown in Fig. 2. (A, B) Levels of hypothalamus CGRP protein expression (pg/ml) in male and female mice respectively. (C) Comparison of hypothalamus CGRP protein expression (pg/ml) between male and female mice. (D, E) Levels of hypothalamus PACAP protein expression (pg/ml) in male and female mice respectively. (F) Comparison of hypothalamus PACAP protein expression (pg/ml) between male and female mice. Data were presented as mean ± SEM. One-way ANOVA was used for comparison among the three groups and two-tailed unpaired t-test was used for comparison between male and female group. Significance differences were indicated as * P < 0.05, *** P < 0.001, or **** P < 0.0001

Fig. 5

RNA-sequencing and enrichment analysis revealed profound alternation of transcriptome of TG in male photophobia mice sensitive to pre-treatment of SRCT. The merged TG of each mouse was collected from the same male mouse cohort as the light aversion behavior shown in Fig. 2. (A) Venn diagram visualized numbers of differentially expression genes (DEGs, adjusted P-value < 0.05) identified from UMB vs. DMSO and UMB_SRCT vs. UMB groups. The overlapped region showed the number of DEGs that are sensitive to SRCT. (B) Heatmap showed the expression level of 1067 DEGs in each sample of the three experimental groups. 95.5% of these DEGs were downregulated by UMB, of which 98.0% were restored by pre-treatment of SRCT. (C, D) The volcano plot depicted the numbers of DEGs (|log2FoldChange|≥0, adjusted P-value ≤ 0.05) between UMB vs. DMSO and UMB_SRCT vs. UMB. Each dot represents a DEG, and red or blue dots indicate down- or up- regulation, respectively; The transcripts with |log2FoldChange|>20 in both two comparisons were labelled by the names of genes they transcript from. (E) Gene ontology enrichment analysis and the top 20 terms in the categories of biological process (BP) for the SFKs-sensitive DEGs in male photophobia mice (adjusted P-value < 0.05) and (F) KEGG pathway analysis and the top 20 pathways for the SFKs-sensitive DEGs in male photophobia mice. The count represents numbers of DEGs enriched to respective top 20 functions or pathways

Fig. 6

Differential gene expression of selected candidate genes of TG in male mice in control, photophobia, and photophobia with SRCT groups. The merged TG of each mouse was collected from the same male mouse cohort as the light aversion behavior shown in Fig. 2. Gene level counts were calculated by DEseq2 package and its functions. Normalized counts were compared between control (n = 7, red) vs. photophobia (n = 7, purple) groups and photophobia vs. photophoibia + SRCT (n = 7, blue) groups using the Wald test for significance. Changes in gene expression of (A) Crebbp, (B) Trpm3, (C) Zmynd8, (D) Akt1, (E) Atp5a1 and (F) Scn8a were presented in respective order. Mouse number and sex are indicated. Group data were presented as mean ± SEM. Significant differences were indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001

Fig. 7

The photophobia-induced expression of the selected candidate genes of TG showed gender-different modulation by SFKs antagonism assessed by qPCR. The merged TG of each mouse was collected from the same male and female mouse cohort as the light aversion behavior shown in Fig. 2. The relative gene expression levels in control (red, male n = 6 or 7, female n = 12), photophobia (purple, male n = 6 or 7, female n = 12) and photophobia + SRCT (blue, male n = 6 or 7, female n = 11 or 12) groups were quantified using qPCR and presented by the fold changes normalized to the geometric mean of peptidylprolyl isomerase A (PPIA) and β-actin (ACTB), and expressions were determined using 2^−ΔCT method. Changes in gene expression level of (A) Crebbp, (B) Trpm3, (C) Zmynd8, (D) Akt1, (E) Atp5a1 and (F) Scn8a between control vs. photophobia groups, photophobia vs. photophoibia + SRCT groups, and male vs. female were presented in respective order. Either Mann-Whitney test (abnormal distributed data) or unpaired t-test (normally distributed data), one-tailed test was used for significance. Group data were presented as mean ± SEM. Significant differences were indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001. * Comparisons within the same gender and # comparisons between male and female