Pou4f3 gene mutation promotes autophagy and apoptosis of cochlear hair cells in cisplatin-induced deafness mice.

Pou4f3 plays an important role in the development of hair cells in the inner ear sensory epithelia. Autophagy is related to the auditory damage. However, the role and mechanism of Pou4f3 on drug-induced ototoxicity are incompletely understood. Hence, this study aimed to explore the effects of Pou4f3 on the apoptosis of cochlear hair cells (CHCs) and to explore whether autophagy was involved in this process. The cisplatin was used to produce a loss of CHCs to create a murine model of deafness. The AAV vectors were delivered into the scala media through the lateral wall. Compared with the control mice, the cisplatin-treated mice exhibited significantly enhanced apoptosis and autophagy in the cochleae, accompanied by a notably decreased Pou4f3 levels. Both mutation and knockdown of Pou4f3 promoted the apoptosis- and autophagy-related protein levels, and enhanced the cisplatin-induced levels of apoptosis- and autophagy-related proteins. Furthermore, the autophagy activator rapamycin promoted the apoptosis and autophagy in the cochlea. In addition, the autophagy inhibitor 3-MA overturned the promoting effect of Pou4f3 knockdown on the apoptosis and autophagy. Collectively, in cisplatin-induced deafness mice, the Pou4f3 gene mutation facilitated apoptosis of cochlear hair cells, at least partially, through inducing autophagy.

Fasudil attenuates glial cell-mediated neuroinflammation via ERK1/2 and AKT signaling pathways after optic nerve crush.

To investigate the functional role of fasudil in optic nerve crush (ONC), and further explore its possible molecular mechanism. After ONC injury, the rats were injected intraperitoneally either with fasudil or normal saline once a day until euthanized. RGCs survival was assessed by retrograde labeling with FluoroGold. Retinal glial cells activation and population changes (GFAP, iba-1) were measured by immunofluorescence. The expressions of cleaved caspase 3 and 9, p-ERK1/2 and p-AKT were detected by western blot. The levels of the pro-inflammatory cytokines were determined using real-time polymerase chain reaction. Fasudil treatment inhibited RGCs apoptosis and reduced RGCs loss demonstrated by the decreased apoptosis-associated proteins expression and the increased fluorogold labeling of RGCs after ONC, respectively. In addition, the ONC + fasudil group compared had a significantly lower expression of GFAP and iba1 compared with the ONC group. The levels of pro-inflammatory cytokines were significantly reduced in the ONC + fasudil group than in the ONC group. Furthermore, the phosphorylation levels of ERK1/2 and AKT (p-ERK1/2 and p-AKT) were obviously elevated by the fasudil treatment. Our study demonstrated that fasudil attenuated glial cell-mediated neuroinflammation by up-regulating the ERK1/2 and AKT signaling pathways in rats ONC models. We conclude that fasudil may be a novel treatment for traumatic optic neuropathy.

Up-regulation of autophagy and apoptosis of cochlear hair cells in mouse models for deafness.

INTRODUCTION: Hearing loss is one of the most common sensory disorders. Recent findings have shown that the apoptotic program and autophagy are related to hearing loss. The aim of the study was to explore the effects of noise and cisplatin exposure on apoptosis and autophagy in the hair cells of the cochleae. MATERIAL AND METHODS: C57BL/6 mice were randomly divided into 3 groups (n = 10 for each): the control group, the noise model group and the cisplatin model group. Auditory brainstem response (ABR) measurements were used to detect the hearing thresholds. TUNEL assay was used to evaluate cell apoptosis. Western blot and immunofluorescence were performed to examine the apoptosis- and autophagy-related proteins. RESULTS: The mice exhibited substantial hearing loss after noise and cisplatin exposure. Additionally, more TUNEL positive cells were observed in the mice after noise and cisplatin exposure compared with the control group. Moreover, the protein expression levels of Beclin-1, LC3-II, Bax and cleaved caspase-3 were significantly increased, while the expression of Bcl-2 was notably decreased in the cochlea after noise (p = 0.0278, 0.0075, 0.0142, 0.0158, 0.0131 respectively) and cisplatin (p = 0.0220, 0.0075, 0.0024, 0.0161, 0.0452 respectively) exposure compared with the control group. Besides, the ratio of LC3-II/LC3-I was substantially higher in the mice treated by cisplatin (p = 0.0046) and noise (p = 0.0220) compared with the control group. CONCLUSIONS: Our findings demonstrated for the first time that noise and cisplatin exposure promoted apoptosis and autophagy in the hair cells of the cochleae. This study provides new insights into the mechanisms of noise- or cisplatin-induced hearing loss.

Association of High-Mobility Group Box-1 with Inflammationrelated Cytokines in the Aqueous Humor with Acute Primary Angle-Closure Eyes.

AIM: The aim of this study was to measure the levels of High-mobility group box-1 (HMGB1) and inflammation-related cytokines in the aqueous humor of patients with acute primary angle-closure glaucoma (APAG) and age-related cataract eyes (ARC). METHODS: Aqueous humor samples were obtained from 59 eyes of 59 Chinese subjects (APAG, 32 eyes; and ARC, 27eyes). The multiplex bead immunoassay technique was used to measure the levels of HMGB1 and IL-8, IL-6, G-CSF, MCP-3, VEGF, sVEGFR- 1, sVEFGR-2, TNF-α, PDGF, and IL-10 in aqueous. The data of Patients' demographics and preoperative intraocular pressure (IOP) were also collected for detailed analysis. RESULTS: The APAG group showed significantly elevated concentrations of HMGB1, IL- 8, IL-6, G-CSF, VEGF, sVEGFR-1, and TNF-α than those in the ARC group. Aqueous HMGB1 level correlated significantly with IOP, IL-8, IL-6, G-CSF and sVEGFR-1 levels but not with age, TNF-α, or VEGF levels. CONCLUSION: The aqueous level of HMGB1 is elevated in APAG and associated with aqueous level of inflammation-related cytokines, suggesting an association between elevated levels of HMGB1, APAC and certain inflammatory modulators which, of course, should lead to further investigations in order to demonstrate the cause and effect.

The expression and role of PIDD in retina after optic nerve crush.

To examine the expression of P53-induced protein with a death domain (PIDD) at retina in animal model of optic nerve crush (ONC) and to investigate the role of PIDD in retinal glial activation and NF-κB activation induced by optic nerve damage, ONC animal model was established in Sprague-Dawley rats. PIDD has three isoforms (Isof); Western blot was performed to examine the expression of PIDD (Isof-1, Isof-2, and Isof-3, respectively) in retina at different time points after ONC. Retinal glial activation is closely associated with retinal neuronal death and is monitored by the expression of GFAP+ glial cells and IBA1+ microglia, then activated microglia leads to inflammatory cytokine production. NF-kB activation in glial cells also can promote neuronal death. In our study, the role of PIDD in retinal glial activation and NF-kB activation was investigated with PIDD inhibition selectively. PIDD expression (Isof-1 and Isof-3) was dramatically increased, and peaked at 3 days after ONC, while Isof-2 did not show any difference. In the ONC animal model, the number of GFAP+ glial cells and IBA1+ microglia in retinal layers was increased significantly, inflammatory cytokine production was upregulated, and NF-κB in glial cell was also activated. Moreover, those responses induced by optic nerve damage were attenuated with PIDD inhibition, which indicated that PIDD could regulate retinal glial activation, neuro-inflammation, and NF-κB activation. These results provided the direct demonstration that the PIDD (Isof-1and Isof-3) was overexpressed in retina after ONC, and PIDD may be involved in retinal neurodegenerative diseases by regulating retinal glial activation and NF-κB activation.

Involvement of Upregulated P53-Induced Death Domain Protein in Retinal Ganglion Cells Apoptosis After Optic Nerve Crush.

PURPOSE: Retinal ganglion cells (RGCs) apoptosis is a common characteristic of optic neuropathies. p53-induced protein with a death domain (PIDD) is a well-known regulator of genotoxic stress-induced apoptosis, which is constitutively cleaved into three main fragments: PIDD-N, PIDD-C and PIDD-CC. Thus, we aim to determine the physiological relevance of PIDD in RGCs apoptosis in an optic nerve crush (ONC) model. METHODS: All animals were evenly randomized into four groups: sham-control group, con-siRNA group, ONC group, and PIDD-siRNA group (ONC +PIDD-siRNA). Expressions of PIDD, caspase-2, Brn3a and tBid in ONC model were analyzed by Western blot and immunofluorescence. Mean densities of RGCs/mm2 were calculated with Fluoro-Gold (FG). Moreover, we tested the effect of PIDD-siRNA on ONC-induced RGCs apoptosis using TUNEL staining. RESULTS: The level of full-length PIDD was weakly present and showed no significant differences at any time points. PIDD-CC and PIDD-C were significantly up-regulated in the retina at 3 days after ONC. Meanwhile, the expression of PIDD was significantly increased in Brn3a (a marker of RGCs) positive cells, indicating that the localization of PIDD appeared to be confined to RGCs. Furthermore, inhibition of PIDD prevented RGCs apoptosis by inhibiting caspase-2 and tBid activation. CONCLUSION: Taken together, PIDD may play a crucial role in RGCs apoptosis after ONC, and this process may be relevant to caspase-2 and tBid.