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Dync1li1 is required for the survival of mammalian cochlear hair cells by regulating the transportation of autophagosomes [1]

['Yuan Zhang', 'Department Of Otolaryngology Head', 'Neck Surgery', 'Affiliated Drum Tower Hospital Of Nanjing University Medical School', 'Jiangsu Provincial Key Medical Discipline', 'Laboratory', 'Nanjing', 'Research Institute Of Otolaryngology', 'Shasha Zhang', 'State Key Laboratory Of Bioelectronics']

Date: 2022-08 

(A, B) Dync1li1 mRNA (A) and protein (B) expression in P3 mouse cochlea by RT-PCR and Western blotting, respectively. Brain tissue and the HEI-OC1 cell line were used as the positive controls. (C) Whole mount immunofluorescent staining of Dync1li1 in P3 mouse cochlea. The large square image is a single XY slice, the vertical red line shows the position of the orthogonal slice, which is shown on the right side of each panel, and the blue line on the orthogonal line shows the level of the XY slice on the left. (D) Frozen section immunofluorescent staining of Dync1li1 in P3 mouse cochlea. The white boxes and the dotted lines show enlarged images. (E) Whole mount immunofluorescent staining of Dync1li1 in P30 adult mouse cochlea. For all experiments, scale bars are shown in the figure. (F) A diagram of the IHC and OHC in the cochlea.

RT-PCR showed that Dync1li1 mRNA was highly expressed in postnatal day 3 (P3) mouse cochlea and in the HEI-OC1 cell line ( Fig 1A ). Dync1li1 protein was also detected by western blotting in P3 cochlea ( Fig 1B ). Next, immunofluorescent staining showed that Dync1li1 was highly expressed in HCs in both whole mount and frozen sections of P3 (newborn) and P30 (adult) mouse cochlea. In addition, we also observed that Dync1li1 is also expressed in other types of cells in the organ of Corti, such as spiral neurons and some of supporting cells ( Fig 1C–1E ). Fig 1F shows a diagram of the IHC and OHC of cochlea. These results suggested that Dync1li1 is highly expressed in HCs and might play important roles in HCs in both the neonatal and adult cochlea.

(A) TUNEL assay of P21 and P30 Dync1li1 KO mice and WT control mice. Myo7a and DAPI were used as HC and nuclear markers, respectively. Apoptotic cells are indicated by green arrows. (B) Quantification of the mRNA expression of apoptosis related genes in the cochlea of P30 Dync1li1 KO mice and the control mice by qPCR. Red boxes indicate the genes with significant expression differences. For all experiments, scale bars and N number are shown in the figure. **p < 0.01.

TUNEL signals, which are indicative of apoptosis, were observed in HCs of both P21 and P30 Dync1li1 KO mice, but not in the control group ( Fig 3A ). Real-time qPCR results also showed that the expression of apoptosis-related genes, such as Aparf and Caspase3, were significantly upregulated in the Dync1li KO mouse cochlea ( Fig 3B ). Together, these results indicate that HC loss in Dync1li1 KO mice was due to HC apoptosis.

(A) Western blotting of Dync1li1 in P30 mouse cochlea. Gapdh was used as the internal reference. (B) OHC loss (indicated by asterisks) is seen in the apical (APEX), middle (MID), and basal (BASE) turns of P21, P30, and P60 Dync1li1 KO and wild-type (WT) mice cochlea. Myo7a (red) was used as the HC marker. (C, D) Quantification of the OHCs (C) and IHCs (D) in the apical, middle, and basal turns of P21, P30, and P60 Dync1li1 KO and WT mice cochlea. (E, F) Hair bundles were observed by immunofluorescent staining of phalloidin (E) and scanning electron microscopy (F). The enlarged images in the white box in (E) is shown in the lower left corner. (G) The ABR hearing test of Dync1li1 KO mice and control mice at P21, P30, and P60. For all experiments, scale bars and N number are shown in the figure. *p < 0.05, **p < 0.01, n.s. not significant.

Next, we showed that Dync1li1 is indeed knocked out in Dync1li1 KO mice (Figs 2A and S4 ). We first sacrificed mice from neonatal to adult ages to investigate whether HC number is affected by Dync1li1 KO. We saw no significant HC loss before P21 in Dync1li1 KO mice, while slight HC loss could be observed from P21 and HC loss gradually became more and more severe as the mice aged (Figs 2B and S1 ). Quantification of HC loss showed no significant HC loss in P21 mice, although a few HCs were lost in the basal turn. However, significant OHC loss was seen in the apical, middle, and basal turns of the cochlea in P30 and P60 mice ( Fig 2C ), while the number of IHCs was not significantly changed ( Fig 2D ). Immunofluorescent staining and scanning electron microscopy (SEM) both showed that the hair bundles of surviving HCs in Dync1li1 KO mice had normal morphology ( Fig 2E and 2F ). Because HCs are important sound-sensing cells, we used auditory brainstem response to detect the hearing ability of Dync1li1 KO mice. Consistent with the degree of HC loss, the hearing thresholds of the Dync1li1 KO mice were not affected at P21 but were significantly increased at P30 and P60 ( Fig 2G ). Together these results suggest that Dync1li1 is highly expressed in HCs and that its deletion results in progressive HC loss and hearing loss in adult mice, and thus that Dync1li1 plays important roles in HC survival.

Dync1li1 KO decrease the stability of Dynein complex in HCs

Because Dync1li1 is an important part of the dynein complex, which is crucial in all eukaryotic cells for transporting a variety of essential cargoes toward the minus end of microtubules (also called retrograde transport), we speculated that HC apoptosis caused by Dync1li1 KO might be related to impaired retrograde transportation. Thus, we first detected the expression of other components of the dynein complex to determine the stability of dynein. The mRNA levels of Dync1h1 (DHC), Dync1i1 (one of the DIC genes), and Dync1l1 (one of the DLC genes) were all significantly downregulated in P60 Dync1li1 KO mouse cochlea (Fig 4A), and the protein level of Dync1i1/2 (DIC) and Dyncll1 (DLC) were also significantly downregulated, with Dync1i1/2 being the most pronounced and the expression of Dync1li2 was not significantly changed in P30 Dync1li1 KO mice (Fig 4B and 4C). We then used TEM to explore the effects on transport-related organelles in cochlear HCs, such as the structure of the endoplasmic reticulum (ER) and the Golgi apparatus, and we found that the number of lamellae per Golgi was significantly reduced in Dync1li1 KO OHCs compared to the control group and that the Golgi apparatus was thinner in the Dync1li1 KO OHCs (Fig 4D–4F). Together, these results indicate that deletion of Dync1li1 leads to unstable dynein complexes and to decreased stacks of Golgi apparatus in cochlear OHCs.

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TIFF original image Download: Fig 4. Dync1li1 deficiency affect the integrity of Dynein complex and Golgi apparatus. (A) Quantification of the mRNA expression of important subunits in dynein complex (Dync1h1, Dync1i1, and Dynll1) in P60 Dync1li1 KO and the control mice by qPCR. N = 3. N refers to 3 independent qPCR experiments were performed. (B, C) Western blotting (B) and quantification of the western blotting (C) of the Dynein subunit in the cochlea of P60 Dync1li1 KO mice. Gapdh was used as the internal reference. N is indicated in the figure. (D, E) TEM of OHCs in P60 Dync1li1 KO and control mice. The Golgi apparatus is indicated by red arrows in (D) and red lines in (E). (F) Quantification of the number of lamellae per Golgi. For all experiments, scale bars are shown on the figure and N is indicated in the figure. *p < 0.05, ***p < 0.001. https://doi.org/10.1371/journal.pgen.1010232.g004

HC apoptosis caused by Dync1li1 KO is due to impaired transportation of autophagosomes to lysosomes Considering that dynein plays crucial roles during the autophagic process (Fig 5D) and that autophagy is involved in apoptosis [46], we hypothesized that the HC apoptosis caused by Dync1li1 KO is due to impaired transportation of autophagosomes to lysosomes. To test this, we first detected the LC3 signal (autophagic vacuoles marker) in HCs of P60 Dync1li1−/−LC3-GFP mice and found significantly more LC3-positive puncta in the OHCs of Dync1li1−/−LC3-GFP mice compared to the LC3-GFP–only control mice (Fig 5A and 5B). Western blotting showed that the LC3 and Sqstm1/p62 protein level were upregulated in P60 Dync1li1 KO mouse cochlea, suggesting that autophagic vacuoles were aggregated in the HCs (Fig 5C). Moreover, Rab7, an adaptor protein during the maturation of autolysosomes [47,48], was also upregulated in the cochlea of P60 Dync1li1 KO mice (Fig 5C), which indicated that newly formed autophagosomes (with Rab7 on their surface) could not be transported to lysosomes for degradation and thus had accumulated in the HCs.

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TIFF original image Download: Fig 5. Dync1li1 deficiency induced the accumulation of autophagosomes vacuoles in HCs. (A) Immunofluorescence of LC3 (green puncta, indicated by white arrows) in HCs of P60 Dync1li1−/−LC3-GFP mice and control mice. Myo7a was used as HC marker. The enlarged images are shown in the lower left corner. (B) Quantification of the number of LC3+ OHCs. N = 3. (C) Western blotting of the LC3 (LC3-I, 16 kDa; LC3-II, 14 kDa.), Rab7, and Sqstm1/p62 in the cochlea. Gapdh was used as the internal reference. (D) Schematic of the role of dynein in mediating autophagosome–lysosome fusion. Rab links autophagosomes to dynein to mediate microtubule-dependent minus-end-directed transportation towards the lysosome. For all experiments, scale bars are shown in the figure. *p < 0.05, **p < 0.01. https://doi.org/10.1371/journal.pgen.1010232.g005

Next, we verified these results in the HC-like HEI-OC1 cell line [49]. We used shRNA to knock down Dync1li1 in the HEI-OC1 cell line and confirmed the KD efficiency (Fig 6A and 6B). We then transfected the LC3-RFP plasmid into HEI-OC1 cells and quantified the LC3 puncta, and we found that the number of LC3 puncta was significantly increased in the Dync1li1 KD group at both 24 h and 36 h after transfection compared to the controls (Fig 6C and 6D).

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TIFF original image Download: Fig 6. Accumulation of autophagosomes in Dync1li1 KD HEI-OC1 cells. (A, B) OC1 Cells were transfected with shDync1li1-GFP shRNA, and qPCR analysis (A) and Western blotting (B) were used to test the knockdown efficiency of Dync1li1. shNC-GFP was used as control shRNA. The cells were harvest after 36h transfection. (C, D) shNC-GFP and shDync1li1-GFP were cotransfected with LC3-RFP plasmids into OC1 cells for 24 h and 36 h, respectively. Red dots in (C) indicate the LC3 puncta (autophagic vacuoles), which were quantified in (D). (E) Schematic of the working principle of the RFP-GFP-LC3 plasmid. In a neutral environment (autophagosome), LC3 is expressed with both GFP and RFP fluorescencent proteins, and thus the autophagosome dots (RFP+GFP+) are yellow. In an acidic environment (autolysosome), GFP fluorescence is quenched, and thus the autolysosome dots (RFP+) are red. (F, G) siNC and siDync1li1 siRNA were cotransfected with RFP-GFP-LC3 plasmid for 36 h. Yellow arrow heads and red arrows in (F) indicate the autophagosomes (yellow dots) and the autolysosomes (red dots), respectively. The enlarged images are shown in the upper left in (F). The number of autophagosomes and autolysosomes in the Dync1li1 KD cells and the control cells were quantified in (G). (H, I) siNC and siDync1li1 siRNA were transfected for 24 h and 36 h, respectively. Western blotting (H) and quantification of Dynein subunit proteins after siRNA transfection. Quantification of protein expression levels at 36 h after siRNA transfection (I). (J) Western blotting of LC3, Sqstm1/p62 and Rab7 at 36 h after siRNA transfection. For all experiments, scale bars are shown in the figure. *p < 0.05, **p < 0.01, n.s. not significant. https://doi.org/10.1371/journal.pgen.1010232.g006

RFP-GFP-LC3 is a tool plasmid for detecting the level of autophagy in cells as illustrated in Fig 6E [50]. There are two fluorescent protein, red RFP and green GFP, expressed as a fusion protein with LC3, in which GFP is a pH sensitive protein. When in the autophagic vacuoles with a neutral pH (autophagosome), RFP and GFP both show fluorescent signal, and thus the LC3 dots are yellow (GFP+/RFP+). When in the autophagic vacuoles with an acidic pH (autolysosome), GFP cannot show fluorescent signal and only RFP can show red fluorescent signal, and thus the LC3 dots are red. Therefore, we can use this plasmid to measure autophagic flux in the Dync1li1 KD group and the control group. And we found that in Dync1li1 KD OC1 cells, the number of autophagosomes (GFP+/RFP+) were significantly more than that in the control group, while the number of autolysosomes (GFP-/RFP+) was less than that in the control group (Fig 6F and 6G). These data suggested that Dync1li1 KD led to accumulation of autophagosomes which cannot be eliminated by transporting to lysosomes to form autolysosomes. Moreover, we also found that the protein level of Dync1i1/2 (DIC) and Dyncll1 (DLC) were also significantly down regulated, the expression of Dync1li2 was not changed, and the protein level of LC3 and Sqstm1/p62 were up regulated in Dync1li1 KD group (Fig 6H and 6I). These results are consistent with the changes in protein level in Dync1li1 KO mouse cochlea. Therefore, we conclude that knock down of Dync1li1 in vitro led to impaired transportation of autophagosomes to lysosomes and to abnormal accumulation of autophagosomes in HC-like HEI-OC1 cells.

In summary, the mechanisms we identified are shown in Fig 7. Under normal conditions, late autophagosomes with harmful substances produced in HCs are transported by dynein to be fused with lysosomes and form autolysosomes for subsequent degradation, thereby maintaining cell homeostasis. When Dync1li1 is defective or missing, the dynein complex becomes unstable and cannot effectively transport late autophagosomes to lysosome for degradation, which leads to the accumulation of these autophagosomes with harmful substances. This disruption in cell homeostasis triggers HC apoptosis and thus leads to hearing loss.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010232

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