The effect and mechanism of 19S proteasome PSMD11/Rpn6 subunit in D-Galactose induced mimetic aging models

https://doi.org/10.1016/j.yexcr.2020.112093Get rights and content

Highlights

  • D-Galactose (D-gal) can be used to induce advanced degeneration models for presbycusis in vivo and in vitro.

  • The function of proteasome is impaired with aging in D-gal mimetic aging models.

  • PSMD11 plays a vital role in protecting presbycusis through interaction with AMPKa2, but not a1, in D-gal induced mimetic aging models.

Abstract

Regulating proteasome activity is a potent therapeutic aspect of age-related hearing loss, which has been proven to protect neurons from age-related damaging. PSMD11, subunit of the 19S proteasome regulatory particle, is known to mainly up-regulate proteasome activity and prolong aging. However, the mechanism of PSMD11 in age-related hearing loss has not been deeply explored. In the present study, we explore the function and mechanism of PSMD11 protecting neurons in d-Galactose (D-Gal) mimetic aging models. Age-related pathologies were detected by Taq-PCR, ABR, Transmission electron microscopy, toluidine blue and β-galactosidase staining. The relative expressions of the proteins were explored by Western blotting, oxyblot, immunoprecipitation and immunofluorescence. Flow cytometry was used to manifest the oxidative state. We discovered that proteasome activity was impaired with aging, and that ROS and toxic protein accumulated in D-Gal induced aging models. PSMD11 changed with aging, and was associated with the metabolism of proteasome activity in the D-Gal treated models. Moreover, the knockdown or overexpression of PSMD11 was sufficient to change the oxidative state caused by D-Gal. Our results also demonstrated that PSMD11 could bond to AMPKα1/2 in the auditory cortex and PC12 cells, and AMPKα2 but not AMPKα1 was efficient to regulate the function of PSMD11. Deeper insights into the mechanisms of regulating PSMD11 for the anti-aging process are needed, and may offer novel therapeutic methods for central presbycusis.

Introduction

Aging is an irresistible process for human beings. However, age-related diseases can be modulated and prevented by the progress of medical research. Presbycusis or age-related hearing loss (ARHL), is characterized by age-related bilateral, progressive and inevitable decline of hearing, as well as cognition declination [1,2]. Presbycusis is among the most prevalent chronic diseases in elderly people, thirdly to hypertension and arthritis. About 25%~40% of people over 65 years old suffer from presbycusis, concomitant loneliness, depression and poor health status, which seriously impact quality of life among elderly people [[3], [4], [5]]. Studies on the central auditory system till now have mainly focused on mitochondrial dysfunction with abnormal ROS generation [[6], [7], [8], [9], [10]], accelerated ROS damage, mtDNA mutations and neuron cell apoptosis [[11], [12], [13]]. The mechanism of the central auditory system involved in cognition is not fully understood, which offers a challenge to treat short-term memory and auditory perception in aging.

Even though molecular theories of ROS and mitochondrial dysfunction have been demonstrated in aging and central ARHL, our knowledge about proteasome associated proteolysis dysfunction in the central auditory system is limited. The 26S proteasome composed with 20S CP (proteolytic core particle) and 19S RP (regulatory particle) and plays a major role in maintaining the balance of protein degradation [14,15], which should assemble and behave accordingly to exert its function of removing wasted, misfolded and toxic proteins [16,17]. The proteasome abundance is regulated at the level of proteasome subunits assembly, and the proteasome activity is related to some subunits of 19S RP [18,19]. Further, the 19S RP, containing two sub-complexes located on the lid and base of 20S CP, is responsible for recognizing, deubiquitinating and transferring substrates into 20S CP for degrading [16]. Depending on its structure and function, 19S RP is the most important rate-limiting portion of the UPS [20,21]. In pre-mature aging, moderate oxidative stress stimulates 19S RP, which activates proteasome expression and stimulates the ability of proteasome to degrade oxidized proteins. However, the reduction of 19S proteasome subunits, like Rpn6, Rpn10, Rpt2 and Rpt6, accompanied by impaired proteasome activity gives rise to central aging and neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, and Alzheimer's disease [[22], [23], [24], [25], [26]]. Until present, few studies have discussed the mechanisms for the subunits of the 19S proteasome RP in ARHL, so regulating proteasome activity is a new frontier in the study of anti-central ARHL [20,21].

Among the studies on 19S RP, the PSMD11, which is homologous to Rpn6, is prominent in prolonging life-span by increasing 26S proteasome assemble and activity in the C. elegans model [[27], [28], [29]]. In the study of stem cell differentiation, PSMD11 increases proteasome degradation activity and prevents the effects of multiple proteome threats like heat, UV and oxidative stress under the control of FOXO4 in human embryonic stem cells (hESCs) [30,31]. Moreover, overexpression of PSMD11 activates the proteasome functions and degradation of some aggregation-prone proteins under the control of cAMP/PKA regulation [32]. Only a few studies, however, mentioned the reduction of PSMD11 in aging and neurodegenerative diseases without further study, left alone in the ARHL. Moreover, studies on the other 19S proteasome subunits, such as Rpt2 and Rpt6, suggest that the 19S proteasome is under metabolic control [33,34]. However the regulation of proteometabolism disorder in the central presbycusis directly through the 19S proteasome subunit is limited.

d-Galactose (D-Gal) can induce neural damage and cognitive dysfunction in the auditory cortex, leading to central presbycusis. D-Gal has been widely used in studies on aging and age-related diseases [[35], [36], [37]]. Additionally, the accumulation of mtDNA common deletion (CD) [[38], [39], [40]] has been replicated in our previous studies as a biomarker of aging in D-Gal mimetic central presbycusis [6,10]. In this study, we explore the function and regulation of the 19S RP subunit PSMD11 in D-Gal mimetic ARHL. A series of molecular techniques were used to detect proteasome impairment in the central ARHL, including the relative expression of PSMD11, ploy-ubiquitinated, oxidative damaged and apoptosis associated proteins after D-Gal induction, and the mutual regulation of PSMD11-AMPKα1/α2 among in -vivo and in -vitro D-Gal mimetic aging models.

Section snippets

CD accumulation and neuron apoptosis

Mutations of mtDNA are widely implicated in senescence and degenerative diseases, which are generated by ROS attack and imbalance of mitochondrial DNA [11,13,41]. Mitochondrial common deletions (mt CD), including deletions in the 4834 -base-pair of rats and the corresponding 4977 -base-pair of humans, are accepted as biomarkers of aging [38,40,42]. The levels of mtDNA CD were determined by TaqMan RT-PCR analysis according to previous studies [43]. As indicated in Fig. 1A, the relative

Discussion

In the present study, we explored underlying mechanisms of the aging process involved in protein homeostasis disorder, oxidative stress and cell apoptosis. We focused on the regulatory role of PSMD11, the 19s non-ATPase proteasome subunit, in protecting these processes and provided a new prospect for treating presbycusis. Among our past studies, we have found a D-Gal induced rat model to mimic age-related hearing loss, which showed increased oxidative stress and cell apoptosis [6,10,63], but

Animals

One hundred and fifty male Wistar rats (4 weeks old) were acquired from the Experimental Animal Centre of Tongji Medical College, Huazhong University of Science and Technology. Procedures were carried out, following previous procedures, to establish a d-Galactose induced aging model [6,10,57]. After two weeks of acclimation, the Wistar rats were randomly grouped into either the normal saline group (n = 75) or the D-Gal treated group (n = 75) and were injected daily with the vehicle (0.9%

Funding

The present research was supported by the National Natural Science Foundation of china (grant no. 81873700), the Major State Basic Research development Program of china (973 Program; grant no. 81230021) and the National Natural Science Foundation of china (grant no. 81400465).

Ethics approval and consent to participate

All experiment procedures were proceed and involved the care of animals according to the guidelines of the care and Use of Laboratory Animals of the National Institutes of Health. The study protocol mentioned above was approved by the committee on the Ethics of Animal Experiments of HUST. The IACUC number is 2279.

Data availability statement

All datasets [GENERATED/ANALYZED] for this study are included in the manuscript files.

CRediT authorship contribution statement

Han Wu: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. Haiying Sun: Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing. Zuhong He: Data curation, Formal analysis, Writing - review & editing. Xi Chen: Data curation, Formal analysis, Writing - review & editing. Yongqin Li: Data curation, Formal analysis, Writing - review & editing. Xueyan Zhao: Data curation, Formal analysis,

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

Not applicable.

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