HSD17B7 is required for Auditory Function by Regulating Cholesterol Synthesis in Sensory Hair Cells

Reviewer #1 (Public review):

Summary:

This study identifies HSD17B7 as a cholesterol biosynthesis gene enriched in sensory hair cells, with demonstrated importance for auditory behavior and potential involvement in mechanotransduction. Using zebrafish knockdown and rescue experiments, the authors show that loss of hsd17b7 reduces cholesterol levels and impairs hearing behavior. They also report a heterozygous nonsense variant in a patient with hearing loss. The gene mutation has a complex and somewhat inconsistent phenotype, appearing to mislocalize, reduce mRNA and protein levels, and alter cholesterol distribution, supporting HSD17B7 as a potential deafness gene.

While the study presents an interesting candidate and highlights an underexplored role for cholesterol in hair cell function, several important claims are insufficiently supported, and the mechanistic interpretations remain somewhat murky.

Strengths:

(1) HSD17B7 is a new candidate deafness gene with plausible biological relevance.

(2) Cross-species RNAseq convincingly shows hair-cell enrichment.

(3) Lipid metabolism, particularly cholesterol homeostasis, is an emerging area of interest in auditory function.

(4) The connection between cholesterol levels and MET is potentially impactful and, if substantiated, would represent a significant advance.

Weaknesses:

(1) The pathogenic mechanism of the E182STOP variant is unclear: The mutant protein presumably does not affect WT protein localization, arguing against a dominant-negative effect. Yet, overexpression of HSD17B7-E182* alone causes toxicity in zebrafish, and it binds and mislocalizes cholesterol in HEI-OC1 cells, suggesting some gain-of-function or toxic effect. In addition, the mRNA of the variant has a low expression level, suggesting nonsense-mediated decay. This complexity and inconsistency need clearer explanation.

(2) The link to human deafness is based on a single heterozygous patient with no syndromic features. Given that nearly all known cholesterol metabolism disorders are syndromic, this raises concerns about causality or specificity. The term "novel deafness gene" is premature without additional cases or segregation data.

(3) The localization of HSD17B7 should be clarified better: In HEI-OC1 cells, HSD17B7 localizes to the ER, as expected. In mouse hair cells, the staining pattern is cytosolic and almost perfectly overlaps with the hair cell marker used, Myo7a. This needs to be discussed. Without KO tissue, HSD17B7 antibody specificity remains uncertain.

Reviewer #2 (Public review):

A summary of what the authors were trying to achieve.

The authors aim to determine whether the gene Hsb17b7 is essential for hair cell function and, if so, to elucidate the underlying mechanism, specifically the HSB17B7 metabolic role in cholesterol biogenesis. They use animal, tissue, or data from zebrafish, mouse, and human patients.

Strengths:

(1) This is the first study of Hsb17b7 in the zebrafish (a previous report identified this gene as a hair cell marker in the mouse utricle).

(2) The authors demonstrate that Hsb17b7 is expressed in hair cells of zebrafish and the mouse cochlea.

(3) In zebrafish larvae, a likely KO of the Hsb17b7 gene causes a mild phenotype in an acoustic/vibrational assay, which also involves a motor response.

(4) In zebrafish larvae, a likely KO of the Hsb17b7 gene causes a mild reduction in lateral line neuromast hair cell number and a mild decrease in the overall mechanotransduction activity of hair cells, assayed with a fluorescent dye entering the mechanotransduction channels.

(5) When HSB17B7 is overexpressed in a cell line, it goes to the ER, and an increase in Cholesterol cytoplasmic puncta is detected. Instead, when a truncated version of HSB17B7 is overexpressed, HSB17B7 forms aggregates that co-localize with cholesterol.

(6) It seems that the level of cholesterol in crista and neuromast hair cells decreases when Hsb17b7 is defective (but see comment below).

Weakness:

(1) The statement that HSD17B7 is "highly" expressed in sensory hair cells in mice and zebrafish seems incorrect for zebrafish:

(a) The data do not support the notion that HSB17B7 is "highly expressed" in zebrafish. Compared to other genes (TMC1, TMIE, and others), the HSB17B7 level of expression in neuromast hair cells is low (Figure 1F), and by extension (Figure 1C), also in all hair cells. This interpretation is in line with the weak detection of an mRNA signal by ISH (Figure 1G I"). On this note, the staining reported in I" does not seem to label the cytoplasm of neuromast hair cells. An antisense probe control, along with a positive control (such as TMC1 or another), is necessary to interpret the ISH signal in the neuromast.

(b) However, this is correct for mouse cochlear hair cells, based on single-cell RNA-seq published databases and immunostaining performed in the study. However, the specificity of the anti-HSD17B7 antibody used in the study (in immunostaining and western blot) is not demonstrated. Additionally, it stains some supporting cells or nerve terminals. Was that expression expected?

(2) A previous report showed that HSD17B7 is expressed in mouse vestibular hair cells by single-cell RNAseq and immunostaining in mice, but it is not cited:

Spatiotemporal dynamics of inner ear sensory and non-sensory cells revealed by single-cell transcriptomics.

Jan TA, Eltawil Y, Ling AH, Chen L, Ellwanger DC, Heller S, Cheng AG.

Cell Rep. 2021 Jul 13;36(2):109358. doi: 10.1016/j.celrep.2021.109358.

(3) Overexpressed HSD17B7-EGFP C-terminal fusion in zebrafish hair cells shows a punctiform signal in the soma but apparently does not stain the hair bundles. One limitation is the consequence of the C-terminal EGFP fusion to HSD17B7 on its function, which is not discussed.

(4) A mutant Zebrafish CRISPR was generated, leading to a truncation after the first 96 aa out of the 340 aa total. It is unclear why the gene editing was not done closer to the ATG. This allele may conserve some function, which is not discussed.

(5) The hsd17b7 mutant allele has a slightly reduced number of genetically labeled hair cells (quantified as a 16% reduction, estimated at 1-2 HC of the 9 HC present per neuromast). On a note, it is unclear what criteria were used to select HC in the picture. Some Brn3C:mGFP positive cells are apparently not included in the quantifications (Figure 2F, Figure 5A).

(6) The authors used FM4-64 staining to evaluate the hair cell mechanotransduction activity indirectly. They found a 40% reduction in labeling intensity in the HCs of the lateral line neuromast. Because the reduction of hair cell number (16%) is inferior to the reduction of FM4-64 staining, the authors argue that it indicates that the defect is primarily affecting the mechanotransduction function rather than the number of HCs. This argument is insufficient. Indeed, a scenario could be that some HC cells died and have been eliminated, while others are also engaged in this path and no longer perform the MET function. The numbers would then match. If single-cell staining can be resolved, one could determine the FM4-64 intensity per cell. It would also be informative to evaluate the potential occurrence of cell death in this mutant. On another note, the current quantification of the FM4-64 fluorescence intensity and its normalization are not described in the methods. More importantly, an independent and more direct experimental assay is needed to confirm this point. For example, using a GCaMP6-T2A-RFP allele for Ca2+ imaging and signal normalization.

(7) The authors used an acoustic startle response to elicit a behavioral response from the larvae and evaluate the "auditory response". They found a significative decrease in the response (movement trajectory, swimming velocity, distance) in the hsd17b7 mutant. The authors conclude that this gene is crucial for the "auditory function in zebrafish".

This is an overstatement:

(a) First, this test is adequate as a screening tool to identify animals that have lost completely the behavioral response to this acoustic and vibrational stimulation, which also involves a motor response. However, additional tests are required to confirm an auditory origin of the defect, such as Auditory Evoked Potential recordings, or for the vestibular function, the Vestibulo-Ocular Reflex.

(b) Secondly, the behavioral defects observed in the mutant compared to the control are significantly different, but the differences are slight, contained within the Standard Deviation (20% for velocity, 25% for distance). To this point, the Figure 2 B and C plots are misleading because their y-axis do not start at 0.

(8) Overexpression of HSD17B7 in cell line HEI-OC1 apparently "significantly increases" the intensity of cholesterol-related signal using a genetically encoded fluorescent sensor (D4H-mCherry). However, the description of this quantification (per cell or per surface area) and the normalization of the fluorescent signal are not provided.

(9) When this experiment is conducted in vivo in zebrafish, a reduction in the "DH4 relative intensity" is detected (same issue with the absence of a detailed method description). However, as the difference is smaller than the standard deviation, this raises questions about the biological relevance of this result.

(10) The authors identified a deaf child as a carrier of a nonsense mutation in HSB17B7, which is predicted to terminate the HSB17B7 protein before the transmembrane domain. However, as no genetic linkage is possible, the causality is not demonstrated.

(11) Previous results obtained from mouse HSD17B7-KO (citation below) are not described in sufficient detail. This is critical because, in this paper, the mouse loss-of-function of HSD17B7 is embryonically lethal, whereas no apparent phenotype was reported in heterozygotes, which are viable and fertile. Therefore, it seems unlikely that heterozygous mice exhibit hearing loss or vestibular defects; however, it would be essential to verify this to support the notion that the truncated allele found in one patient is causal.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Jokela H, Rantakari P, Lamminen T, Strauss L, Ola R, Mutka AL, Gylling H, Miettinen T, Pakarinen P, Sainio K, Poutanen M.
Endocrinology. 2010 Apr;151(4):1884-92. doi: 10.1210/en.2009-0928. Epub 2010 Feb 25.

(12) The authors used this truncated protein in their startle response and FM4-64 assays. First, they show that contrary to the WT version, this truncated form cannot rescue their phenotypes when overexpressed. Secondly, they tested whether this truncated protein could recapitulate the startle reflex and FM4-64 phenotypes of the mutant allele. At the homozygous level (not mentioned by the way), it can apparently do so to a lesser degree than the previous mutant. Again, the differences are within the Standard Deviation of the averages. The authors conclude that this mutation found in humans has a "negative effect" on hearing, which is again not supported by the data.

(13) The authors looked at the distribution of the HSB17B7 in a cell line. The WT version goes to the ER, while the truncated one forms aggregates. An interesting experiment consisted of co-expressing both constructs (Figure S6) to see whether the truncated version would mislocalize the WT version, which could be a mechanism for a dominant phenotype. However, this is not the case.

(14) Through mass spectrometry of HSB17B7 proteins in the cell line, they identified a protein involved in ER retention, RER1. By biochemistry and in a cell line, they show that truncated HSB17B7 prevents the interaction with RER1, which would explain the subcellular localization.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Jokela H, Rantakari P, Lamminen T, Strauss L, Ola R, Mutka AL, Gylling H, Miettinen T, Pakarinen P, Sainio K, Poutanen M.
Endocrinology. 2010 Apr;151(4):1884-92. doi: 10.1210/en.2009-0928. Epub 2010 Feb 25.

(15) Information and specificity validation of the HSB17B7 antibody are not presented. It seems that it is the same used on mice by IF and on zebrafish by Western. If so, the antibody could be used on zebrafish by IF to localize the endogenous protein (not overexpression as done here). Secondly, the specificity of the antibody should be verified on the mutant allele. That would bring confidence that the staining on the mouse is likely specific.