Objectives Age-related hearing loss (ARHL) is much more prevalent with age, affecting not only peripheral but central auditory system. We have previously established an aging model of peripheral auditory system in vitro using cultured cochlear basilar membrane. However, there is no ideal accelerated aging model on central auditory system in vitro. To establish the aging model, auditory cortical neurons (ACNs) were primary cultured and treated with either vehicle or different doses of D-galactose (D-gal). We studied the effect of D-gal on ACNs by evaluating the hallmarks of aging, including cell proliferation, oxidative stress, mitochondrial function, and neuronal apoptosis. Compared with the control group, cell viability was significantly inhibited in the D-gal-treated group in a dose-dependent manner. The production of reactive oxygen species was strongly increased in the D-gal-treated group. Meanwhile, the level of 8-hydroxy-2 '-deoxyguanosine, which is a biomarker of DNA oxidative damage, was even higher in the D-gal-treated group than that in the control group. Conversely, the levels of ATP and mitochondrial membrane potential were notably decreased in the D-gal-treated group contrast to that in the control group. Furthermore, the number of neuronal apoptosis in the D-gal-treated group, compared with that in the control group, was dramatically increased in a dose-dependent approach. Together, our results demonstrate that ACNs treated with D-gal in vitro display senescence characteristics by regulating oxidative stress and apoptosis, indicating accelerated aging model on ACNs are successfully established. And the model provides a promising approach for exploring underlying mechanisms of the ARHL.

Renal cell carcinoma (RCC) is one of the most aggressive urologic cancers, however, the mechanism on supporting RCC carcinogenesis is still not clear. By using gene expression profile analysis and functional clustering, PDZ domain-containing 1 (PDZK1) was revealed to be downregulated in human clear cell renal cell carcinoma (ccRCC) samples, which was also verified in several independent public ccRCC data sets. Using PDZK1 overexpression and knockdown models in ccRCC cell lines, we demonstrated that PDZK1 inhibited cell proliferation, cell cycle G1/S phase transition, cell migration and invasion, indicating a tumor-suppressor role in the development and progression of ccRCC. Our study further demonstrated that PDZK1 inhibited cell proliferation and migration of ccRCC via targeting SHP-1. PDZK1 was further identified to suppress cell proliferation by blocking SHP-1 phosphorylation at Tyr536 via inhibition of the association between SHP-1 and PLCβ3, and then retarding Akt phosphorylation and promoting STAT5 phosphorylation in ccRCC cells. Moreover, the inhibitive effects of PDZK1 on SHP-1 phosphorylation and the tumor growth were verified in vivo by xenograft tumor studies. Accordingly, PDZK1 expression was negatively correlated with SHP-1 activation and phosphorylation, advanced pathologic stage, tumor weight and size, and prognosis of ccRCC patients. These findings have provided first lines of evidences that PDZK1 expression is negatively correlated with SHP-1 activation and poor clinical outcomes in ccRCC. PDZK1 was identified as a novel tumor suppressor in ccRCC by negating SHP-1 activity.

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.

Hyaluronic acid hydrogels modified with polyclonal anti-Nogo-66 receptor antibody were developed in order to promote regeneration in the injured CNS. These modified hydrogels were intended not only to deliver antibodies, but also to serve as a scaffold for neural regeneration following their implantation into injured tissue. Since unmodified hyaluronic acid-hydrogels do not support cell attachment, the gels were modified with polyclonal anti-Nogo-66 receptor with the aim of altering the surface properties of the gels in such a way as to improve neuronal adherence and survival. After evaluating the immobilization efficiency of the system, chicken dorsal root ganglia and dorsal root ganglia cells were planted on the surface of the modified gels to determine cell viability. Dorsal root ganglia were also cultured close to the gels in order to assay the inducement of neurite outgrowth. In dorsal root ganglia and cell viability assay, dorsal root ganglia and neuron cells could adhere to the modified hydrogels and survive well, but it did not happen to unmodified hydrogels. After 72 h, these attached cells were stained positively with immuno-staining for neurofilament. Neurite outgrowth inducement assay showed that the number and length of dorsal root ganglia neurites on the side toward modified hydrogels were significantly more than that on the opposite side (both P<0.01). The results reveal that hyaluronic acid-hydrogels modified with anti-Nogo-66 receptor can support neural cell attachment and survival in vitro. Furthermore, this system can greatly induce neurite outgrowth. The results also indicate that this modified hydrogels have potential to repair injury in the CNS.