The effect of a Pex3 mutation on hearing and lipid content of the inner ear.

Kochaj RM, Martelletti E, Ingham NJ, Buniello A, Sousa BC, Wakelam MJO, Lopez-Clavijo AF, and Steel KP. (2022) The effect of a Pex3 mutation on hearing and lipid content of the inner ear. Cells

Peroxins are encoded by PEX genes and import peroxisomal proteins into a peroxisome, membrane-bound organelles that are essential for lipid metabolism and regulate reactive oxygen species (ROS). Mutations in these PEX genes lead to a spectrum of disorders called peroxisomal biogenesis disorders (PBDs) caused by dysfunctional peroxisomes. One example of a PBD is Zellweger syndrome (ZS) which is characterized by hypotonia, liver and kidney defects, seizures, developmental delays, and can result in early childhood death. The severity of ZS is dependent on the specific mutation expressed, but hearing loss is also a common symptom experienced. Kochaj et al were interested in studying peroxisomes in ZS, and specifically chose to look at how hearing is altered.

To study the role of peroxisomes in ZS, Pex3 mice were chosen since most Pex mutations in mice cause early death, however this is not seen when Pex3 is deficient. As well, in humans the PEX3 gene has been associated with audiometric thresholds at 4 kHz, around the normal audiometric threshold, therefore a mutation in this gene could have a role in hearing loss. Supporting this, the Pex3tm1a mutation causes hearing impairments at high frequencies in mice. To ensure that the mutation prevented transcription of Pex3, Pex3 mRNA levels were measured using quantitative reverse-transcription PCR (qRT-PCR) and they demonstrated that the levels in the homozygous mice were 16% of that seen in wild-type mice while heterozygous animals demonstrated 58% mRNA levels.

To determine if any hearing impairment was occurring in the Pex3tm1a mice, auditory brainstem response (ABR) recording testing was measured using frequencies from 3-42 kHz at levels from 0-95 dB. Mice can start responding to sounds between 12 and 14 days of age so the first measurement was at 17 days to ensure they had met this developmental milestone first. The homozygous mice had slightly increased ABR thresholds suggesting that they experienced relatively normal hearing development. However, by three weeks of age, the Pex3tm1a mutant mice demonstrated increased thresholds compared to the wild-type mice and this continued at four and eight weeks of age. Interestingly, the heterozygous animals had normal ABR threshold levels, indicating that 58% of Pex3tm1a mRNA transcription is adequate for hearing, but 16% is not.

Since the peroxisome is essential for lipid biosynthesis, and specifically the production of a class of lipids called plasmalogens that contain a vinyl-ether bond, a lipidomic analysis was also performed on the inner ear. Plasmalogens are important for cellular membrane fluidity and structure and are necessary for lipid raft formation, vesicular fusion, and cell signaling, so changes to their levels can have distinct effects throughout the body. To determine if an alteration of the inner ear lipidome had a role in the hearing impairment in Pex3tm1a mice, 671 lipid species were measured. Consistently, ether-linked phospholipids and plasmalogens were found to be reduced in the inner ear of mutant mice compared to the wild-type mice, while certain species of sphingomyelin were increased.

Kochaj et al were interested in further understanding the role of peroxisomes in ZS through using the Pex3tm1a mutant line since it has a greater survival rate. Although these homozygous mice do not have a complete knockdown of Pex3, the reduction to 16% of that seen in wild-type animals was still able to elicit the hearing loss phenotype associated with ZS while the 58% of transcription seen in the heterozygous mice did not. Interestingly, this suggests a “dose-response” of the Pex3 alleles where a heterozygous animal shows a mid-level response and a homozygous knockout a nearly full response. Since the peroxisome is the location of plasmalogen biosynthesis, a lipidomic analysis also showed reduced plasmalogen levels in the homozygous mice. In these animals there is a systemic loss of PEX3 activity, as in people with ZS, but other work done by this group demonstrated that PEX3 loss locally within the inner ear is necessary for the hearing impairment, suggesting that a local treatment may be enough for recovery, whether that is through improving peroxisomal activity or increasing plasmalogen levels. As a systemic improvement of either could improve more of the ZS phenotype than just the hearing impairment, further work is needed to determine if a local or systemic method would be a more practical and effective treatment for this symptom of ZS.