Knock out ofKlc2by CRISPR/Cas9 in C57BL/6J mice
Kinesin-mediated cargo transport has always been a research hotspot, but this has mainly focused on neurons26–28, and little research on such transport in the auditory system has been performed. The light chains of kinesins play an important role in the transport processes as they can be connected to different kinds of cargo22–24. Here, we explored whether Klc2 plays a role in the auditory system. First, we used western blot to determine the Klc2 expression pattern in wild-type (WT) mice (Fig. 1A), and we found that Klc2 is widely expressed in various organs. Moreover, the expression level of Klc2 in the cochlea is relatively high, indicating that it might play an essential role in the auditory system. The q-PCR results showed that the cochleae had their highest expression of Klc2 at P14 (Fig. 1B), which is when the development of the organ of Corti is nearly complete. This suggested that Klc2 is related to the maintenance of hearing function. To investigate the specific function of Klc2 in the cochlea, we used CRISPR/Cas9 technology to generate Klc2-null mice. Two specific sgRNAs were designed to target Klc2 between intron 2 and intron 11, which abolishes the whole coding sequence of Klc2. Genomic DNA from mouse pups was extracted for PCR genotyping using the primers shown in Fig. 1C. Potential off-target sites were predicted by an online database (https://cm.jefferson.edu/Off-Spotter/), and every potential off-target site was amplified by PCR followed by sequencing. No off-target sites were found. In the Klc2-null mice, Klc2 could not be detected in the cochlea by q-PCR (Fig. 1D) or by western blot (Fig. 1E), indicating that Klc2 was successfully knocked out.
Klc2 is required for the normal function of the cochlea
Klc2-null mice had a normal gross morphology and did not show any apparent defects compared with their WT littermates. We analyzed the auditory functions of Klc2-null mice as they aged, and at P40 the Klc2-null mice showed significant low-frequency hearing loss (Fig. 2A) while high-frequency hearing was not affected. We performed H&E staining to explore the cochlear morphology of Klc2-null mice (Fig. 2C). The OHCs were lost, but the inner HCs (IHCs), the SGN, and the StV were normal compared to WT mice, which indicated that OHC death was the primary reason for the hearing loss seen in Klc2-null mice. Whole-mount staining of Myo7a and phalloidin, two HC-specific markers, showed that Klc2-null mice lost massive OHCs from the apical region at P40 (Fig. 2B and 2D). Immunofluorescence staining also performed on WT and Klc2-null mice basilar membrane in different time points before P40, and we confirmed that OHCs began to lose at P30 in Klc2-null mice. Thereafter massive cell loss occurred with further age, and the OHCs were nearly completely absent at 8 months of age and hearing was almost completely lost (Figure S1A-C). We further checked the morphology of the stereocilia using SEM (Fig. 3), and no abnormalities were seen in the remaining OHCs, indicating that Klc2 functions mainly in the cell body of OHCs. In addition, we checked the expression of other light chains of kinesin (Klc1, Klc3, and Klc4), and q-PCR of P30 WT and Klc2-null mice showed no significant changes in these three genes (Figure S1D), suggesting that the role of Klc2 in the cochlea is not related to these three genes. In conclusion, Klc2 appears to be essential for the survival of OHCs, and loss of OHCs is the reason behind the hearing loss in Klc2-null mice.
Klc2 Binds To Cuticular Plate-related Proteins
Loss of Klc2 impairs cochlear HC function but does not affect the SGN and StV, so we sought to determine the role that Klc2 plays in HC survival. Klc2 is the light chain of kinesin-1, and it connects to the kinesin heavy chains and to the cargo molecules.
We therefore carried out a co-immunoprecipitation from P30 mouse cochleae using Klc2-specific antibody, followed by protein identification by peptide mass spectrometry (CoIP-MS), to identify the possible cargos that are essential for auditory function in OHCs.
Approximately 916 proteins were identified from the IgG samples and 663 proteins from the Klc2 antibody samples, and 128 proteins were left in the Klc2 antibody samples when the 535 common proteins were subtracted (Figure S2A). As expected, some known Klc2-binding proteins like Kinh and Kif5c were included in these 128 proteins. Classification comparison was performed, and we identified a set of proteins that are reported to be localized to the cuticular plate (Tpm3, Myosin6, Sptb2, and Sptn1) 29–31
. Co-IP were performed to further validate the CoIP-MS result. We chose Myh14, a protein related to noise-induced hearing loss that we have reported on previously, and it showed strong binding affinity for Klc2 in the CoIP-MS data (Figure S2B)32
. However, immunofluorescence of P30 WT and Klc2
-null mice did not show any abnormal morphological structures of spectrin (Figure S2C), which is a well-known marker of the cuticular plate. These results demonstrated that although Klc2 binds to the cuticular plate, it does not affect its morphology.
Knock out ofKlc2disrupts the distribution of mitochondria
TEM was performed to check the subcellular structure of HCs in WT and Klc2-null mice at P30, which was when the hearing loss phenotype first appeared. We found that the structure of the cuticular plate was normal, further indicating that it was not the primary reason for HC death. However, we found that the distribution of mitochondria was affected in the HCs of the cochlea after careful examination. Normally, mitochondria are located along the plasma membrane, under the cuticular plate, and around the nucleus to provide the energy required for OHC activity33, but the mitochondria were concentrated in the center of the cytoplasm of the OHCs from Klc2-null mice (Fig. 4A-C). It is noteworthy that the OHC nuclei of Klc2-null mice were normal at P30 (Fig. 4B), suggesting that there was still a period of time before these cells died, and the abnormal mitochondrial localization might be the primary factor that triggers a series of events that lead to HC death. We concluded that deficiency of Klc2 disrupts the mitochondrial transport system, which depends on kinesin and the microtubule network. The disordered mitochondrial transport system might subsequently affect normal mitochondrial function like energy production, apoptosis, autophagy, etc., thus leading to HC degeneration and hearing loss. Next, to explore the possible cause of OHC death, we co-stained parvalbumin with apoptosis marker caspase-3 and autophagy marker Lc3b, respectively. No positive signal for caspase-3 or Lc3b was observed (Figure S3A). The expression pattern of prestin in Klc2-null mice was normal compared to WT mice (Figure S3B), suggesting that the aberrant mitochondrial distribution did not affect the magnification ability of OHCs. It is reported that inaccurate distribution of mitochondria might cause problems with reactive oxygen species (ROS) production34, but Klc2-null mice did not show this phenomenon (Figure S3C). Interestingly, a decreased number of BK channels was seen in IHCs (Fig. 4E-F), which might be related to the special BK variant (called the DEC splice variant or MitoBK) that is specifically located in mitochondria35–37. In summary, we found that aberrant mitochondrial distribution resulted in HC degeneration, but this was not related to apoptosis, autophagy, or redox imbalance.
Gabaa Receptor Might Be Associated With The Auditory Phenotype
To further explore the intracellular changes caused by defective mitochondrial distribution which leads to HC loss in Klc2-null mice, RNA-seq analysis were performed at P30. We found that 28 genes were up-regulated and 366 genes were down-regulated (Fig. 5A). Moreover, 55 genes were classified as “Transporter activity” (Fig. 5B). From the Gene Ontology (GO) analysis, we found that expression of multiple GABAA receptor family genes was decreased (GABAA receptor subunits α1, β2, α3, δ, α2, α6, and γ1) in the Klc2-null mice (Fig. 5C and 5D). GABA, as one kind of inhibitory neurotransmitter, interacts with GABA receptors in order to regulate cellular activities. In the inner ear, the olivocochlear efferent system is both cholinergic and GABAergic, and several studies have shown that GABA receptor is essential for cochlear function38–40. Liberman constructed a set of mouse models and showed that knockout of three mouse lines (α1, α2, and α6) did not affect cochlea function39. We therefore validated the down-regulated expression of the remaining genes in our RNA-seq results (genes showed in Fig. 5D subtract sunbunits α1, α2, and α6 ) by q-PCR(Fig. 5E). In short, knock out of Klc2 led to the down-regulation of multiple GABAA subunits, which might arise from abnormal mitochondrial localization, and this might be one reason for the disruption of the auditory system.
An indel variant in humanKLC2is associated with low-frequency hearing impairment
A 21-year-old male (case 1707652) complaining bilateral hearing impairment was related to our research, with the onset age of 14 years old. Whole genome sequencing found an indel variant c.1444-8_1444-6delTCC in KLC2 gene (NM_001134775.1) from the patient. This novel variation was not found in the 1000 Genomes Project, ExAC 65000 exome allele frequency data and 1751 ethnicity-matched controls. No family history was complained (Fig. 6A). Otological examinations battery including pure tone audiometric examination, distortion product otoacoustic emission (DPOAE), ABR, cochlear microphonics (CM), speech recognition score (SRS) performed on him illustrated the typical auditory neuropathy. The audiogram of the proband showed low-frequency affected hearing impaired (Fig. 6B) with damaged SRS (20% and 48% for the left and right ear, respectively). DPOAE for the proband was normal (Fig. 6C), and no waves could be detected in ABR testing bilaterally (Fig. 6D). It is worth mentioning that our mouse model also showed low-frequency hearing loss which replicated phenotype in human exactly.
Therapeutic Approaches Using Aav
We next explored therapeutic strategies of hearing impairment caused by Klc2 deficiency. AAV has proved to be a safe and effective viral vectors for genetic therapy in the inner ear41.Hence, we tested whether AAV containing the whole WT Klc2 cDNA sequence could rescue the deafness phenotype in Klc2-null mice. The synthetic vector Anc80L65 was used to viral packaging. At P2, Klc2-null mice were treated with an AAV solution by round window injection41, and saline was injected into littermates as the vehicle control. In addition, we labelled the HA tag and observed high efficiency of infection at P14 (Fig. 7A), and western blot was performed to validate the expression of Klc2. Only the cochlear sample treated with AAV showed a clear band using both anti-HA and anti-Klc2 antibodies (Fig. 7B), indicating that Klc2 was re-expressed only in the cochlea and not in other tissue. We then wanted to know whether re-expressing Klc2 rescues Klc2-null mice from hearing loss. As expected, both the ABR threshold and OHC survival ratio showed an apparent recovery in Klc2-null mice at P40 (Fig. 7C-D), and the mean hearing thresholds at 4 kHz, 8 kHz, and 12 kHz were reduced by about 27 dB (p < 0.001), 34 dB (p < 0.001), and 17 dB (p < 0.05), respectively. We concluded that Klc2 re-expression decreased OHC degeneration and prevented hearing loss, and AAV was an effective vector for Klc2 delievery.