Syntaxin 4 is essential for hearing in human and zebrafish

Abstract

Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1-43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.

Figure 1

Genetic and audiometric data for Pakistani family members with HI due to a splice region variant in STX4. (A) Family 4768 pedigree drawing displaying the segregation of the c.232 + 6T>C STX4 variant. Hearing impaired family members are indicated by a black box (male) or circle (female). Individuals who had a DNA sample that underwent exome sequencing are indicated with an asterisk. (B) STX4 coding isoforms according to GENCODEv39 and location of the splice region variant (indicated with an arrow). (C) Pure-tone audiometry audiograms for hearing impaired family members; x represents the results for the left ear and o for the right ear. Affected individuals show severe-to-profound or profound HI. Ages at which the audiometry was performed: IV:1 [25 years of age], IV:2 (22 year), IV:4 (20 year), IV:5 (21 year), IV:7 (10 year), IV:8 (12 year), IV:9 (14 year) and IV:10 (15 year).

Figure 2

STX4A is expressed in the outer and inner hair cells of the mouse cochlea. Whole mount cochlear tissue from P12 wild-type mice were immunolabeled with anti-syntaxin 4 antibody (green), while phalloidin (red) was used to decorate F-actin cytoskeleton. STX4A (green) was localized in the stereocilia (arrows) of both outer and inner hair cells. Also, STX4A expression was observed in hair cells’ body and cell membrane (arrowheads). Scale bar, 20μm.

Figure 3

Morpholino-based knockdown of stx4 in zebrafish causes deficits of hearing and mechanotransduction. (A) Phenotype assessment based on dosage response for both ATG and SS blockers for stx4. Class severe: defect in eye formation, no tail, small body size and edema; Class moderate: edema and curved body, class mild: curved body and class normal: no visible deformities. (B) ABER in 9ng control injected versus 9ng injected ATG blocker (^**P = 0.0001) and 9ng control injected versus 9ng SS injected morphs (^*P = 0.0017) showing a significant difference. Approximately 9ng ATG vs 9ng SS morphants did not show a significant difference (P = 0.2031). (C) Schematic representation of the zebrafish stx4 gene showing the position of SS morpholino (red line) at the end of exon 3 and start of intron 3. Green line arrows indicate primers used for amplification. The bar diagram shows a scheme of the normal mRNA produced versus SS morpholino injected zebrafish mRNA retaining with exon 3. RT-PCR gel electrophoresis results showing retention of intron 3 between exon 3 and exon 4 in SS morpholino injected embryos. (D) Representative images of 9ng ATG and SS morpholino injected larvae and 9ng control injected larvae at 5 dpf. Severe morphological defects with edema were observed in 9 ng injected larvae as compared to 9ng injected controls. (E) Head to length ratio of 9 ng control injected versus 9ng injected ATG blocker and 9ng control injected versus 9ng SS injected morphs showing a significant difference (^**P = 0.0001). Approximately 9ng ATG versus 9ng SS morphants did not show a significant difference (P = 0.1608) between each other in head to length ratio. (F) Maximum intensity projections of confocal images showing FM1–43 dye uptake in neuromast cells of 9ng ATG and SS morpholino injected larvae vs 9ng control injected larvae at 5 dpf. The right panels show that neuromast cells of stx4-morpholino injected larvae do not uptake FM1–43 dye as compared to 9ng control injected in the left panel. Scale bar: 20μm. All data shown are mean ± SD with two-tailed unpaired Student’s t-test.