Ampk and mtor regulate autophagy through direct phosphorylation of ulk1 pdf

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ampk and mtor regulate autophagy through direct phosphorylation of ulk1 pdf

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Doxorubicin is a commonly used chemotherapeutic agent for the treatment of a range of cancers, but despite its success in improving cancer survival rates, doxorubicin is cardiotoxic and can lead to congestive heart failure. Therapeutic options for this patient group are limited to standard heart failure medications with the only drug specific for doxorubicin cardiotoxicity to reach FDA approval being dexrazoxane, an iron-chelating agent targeting oxidative stress.

Protein Phosphorylation in Human Health. Macroautophagy hereafter called autophagy is an intracellular lysosomal degradation process. Long-lived cytosolic proteins and entire organelles are enveloped by a double membrane. These vesicles are called autophagosomes and are transferred to lysosomes.

Regulation of Autophagy by Protein Phosphorylation

This is an open access article distributed under the terms of Creative Commons Attribution License. Age-related hearing loss ARHL , also known as presbycusis, is a complex degenerative disease characterized by declining auditory function, including increased hearing thresholds and reduced frequency resolution 1. ARHL is one of the most prevalent and chronic conditions of older populations, affecting tens of millions of people worldwide 2 , and may cause social isolation, depression, and even dementia 3.

In addition to degeneration of the peripheral auditory organs, auditory cortex degeneration has been demonstrated to serve a crucial role in ARHL pathogenesis 4 , 5 ; however, the molecular mechanism is not well understood. D-galactose D-gal is a reducing sugar and is oxidized into aldehydes and H 2 O 2 when present at high levels 6. Autophagy is a ubiquitous process that occurs in plant, animal, and fungal cells 13 , and three types of autophagy have been described: Macroautophagy hereafter referred to as autophagy , microautophagy and chaperone-mediated autophagy In mammals, autophagy maintains homeostasis by regulating the degradation of cellular organelles and macromolecules and may be activated by starvation, hypoxia and exposure to toxic agents 13 , 15 — In addition, AMPK promotes activation of autophagy by inhibiting the activity of mTOR complex 1 through phosphorylation of either Raptor or tuberous sclerosis complex 2, which subsequently suppresses the activity of mTOR 22 , Additionally, previous studies have indicated that AMPK- and mTOR-dependent autophagy is involved in neurodegenerative diseases 28 — Therefore, it was hypothesized that AMPK and mTOR are involved in the regulation of autophagy in the degeneration of the auditory cortex in the present model.

However, there were no further studies on this, and whether 4EBP1 regulates autophagy in the degeneration of the auditory cortex is unknown. Thus, this issue was investigated in the present study. Autophagy has been demonstrated to serve important roles in various diseases, including cancer, heart disease, neurodegeneration, autoimmune diseases, aging and infection 31 — At the organismal and cellular levels, autophagy has pro-death or pro-survival functions and has different interactions with apoptosis depending on the context 36 — Nevertheless, to the best of our knowledge, no studies have focused on the role of autophagy and its interactions with apoptosis in central presbycusis.

Therefore, the present study assessed changes of autophagy in the auditory cortex of naturally aging rats and mimetic aging rats by 8 weeks of D-gal injection, and further investigated the role of autophagy and its related mechanism in the degeneration of the auditory cortex. All animal procedures were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health A total of male Sprague Dawley SD rats After the 8-week injection protocol, the rats in the two groups were further divided into a 3-month-old subgroup just after the last injection , a 9-month-old subgroup 6 months after the last injection , and a month-old subgroup 12 months after the last injection , in which the rats were sacrificed at 3, 9 and 15 months, respectively.

The D-Loop region copy was used as the conservative segment. Following perfusion, the brain was dissected from the skull, and the auditory cortex was separated from the brain. Subsequently, the auditory cortex was fixed with the 2. Serial ultrathin sections 60— nm were collected on copper grids and stained at room temperature with uranyl acetate for 30 min and then lead citrate for 10 min. Following a 0.

The labelled cells were observed using a laser scanning confocal microscope Nikon Corporation, Tokyo, Japan , and six fields of view for each section were observed. The primers were designed by Takara Bio, Inc. The protein expression levels were examined using western blotting.

Enhanced chemiluminescent Plus Beyotime Institute of Biotechnology was used to visualize the membranes. Sections that were not incubated with a primary antibody were used as the negative controls.

The statistical analyses were performed using SPSS CDs are also called mt 4,bp deletions and correspond to mtDNA 4,bp deletions in humans Previous research has indicated an association between CD levels and presbycusis In addition, the CDs accumulated with age in both the natural aging group and the mimetic aging group. CD levels increase in D-gal-induced mimetic aging rats. The CD levels in the D-gal-induced mimetic aging rats were higher than those in the age-matched NS subgroups, and the CD levels increased with aging in both groups.

Neurons in the auditory cortex in the NS group did not display obvious ultrastructural changes in the 3- and 9-month-old rats Fig. However, in the month-old rats in the NS group, swollen mitochondria, disrupted myelin and lipofuscin were observed Fig. In the D-gal-treated groups, normal nuclei, mitochondria, Golgi apparatus and rough endoplasmic reticulum were observed in the 3-month-old subgroup Fig.

Additionally, these changes were more pronounced in the month-old D-gal-treated rats Fig. Furthermore, more autophagosomes and auto-lysosomes were observed in the month-old NS group and the 3- and 9-month-old D-gal subgroups, while few autophagosome and auto-lysosomes were identified in the 3- and 9-month-old NS subgroups and month-old D-gal subgroup, particularly in the month-old D-gal subgroup.

Ultrastructural morphology changes with aging in the NS and D-gal groups. Transmission electron microscopy demonstrated ultrastructural changes in the auditory cortex of the NS and D-gal groups at different ages. The red asterisks indicate the mitochondria; the black arrows indicate the disrupted myelin; the yellow arrows indicate the autophagosomes; the red arrows indicate the auto-lysosomes; the white arrows indicate the endoplasmic reticulum; and the black arrowheads indicate the lipofuscin.

Sections were stained with uranyl acetate and lead citrate. Cell apoptosis level is altered at different ages in the auditory cortex in NS- and D-gal-treated rats. It was demonstrated that autophagy was increased from 3 months to 15 months in the NS group, while it decreased with aging in the D-gal group. Aa-c Reverse transcription-quantitative polymerase chain reaction was used to determine the changes in the mRNA expression levels in the D-gal group compared with those in the age-matched NS group.

Furthermore, in addition to LC3 and BECN1, p62 protein expression levels were detected using western blotting and immunofluorescence assays. Additionally, these autophagy-related proteins demonstrated approximately equivalent levels in the 9-month-old D-gal induced mimetic aging group and month-old NS group. Protein expression levels of p62 in the auditory cortex at different ages in NS- and D-gal-treated rats.

D Immunofluorescence assays were used to detect p62 protein expression levels, the nuclei were stained with DAPI and E the levels were quantified. NS, normal saline; D-gal, D-galactose. By investigating the levels of CD, and cell apoptosis and neuronal degeneration at 3, 9 and 15 months in the two groups, the present results demonstrated that the aging level in the month-old rats in the NS group was higher than those in 9-month-old NS subgroup. Additionally, the present results indicated that 9-month-old rats in the D-gal-treated group presented an approximately similar life status compared with that of the month-old rats in NS group, which was further confirmed by immunofluorescence analyses of LC3 and BECN1.

Furthermore, neuronal degeneration and cell apoptosis were more apparent in the month-old D-gal subgroup than the 9-month-old D-gal subgroup. The present results revealed that the function of D-gal exposure that accelerated the aging in rats was confirmed in the present study, which is consistent with previously research 6 , The lifespan of the majority of SD rats is 2.

In other words, rats in the month-old NS subgroup and 9-month-old D-gal subgroup were classified as late adults; month-old rats in the D-gal-induced mimetic aging group demonstrated more severe aging than that of other subgroups and should be classified in the old period. Based on the present results, it was demonstrated that cell apoptosis level increased with aging in the auditory cortex of SD rats, while the level of autophagy showed an increasing trend from young to adult rats and decreased at an old age Fig.

Time-dependent changes in apoptosis and autophagy in the auditory cortex of Sprague Dawley rats. A The trend in cell apoptosis with aging was based on the results of terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling staining. B The trend in autophagy changes with aging was based on the results of the immunofluorescence analysis of the autophagy marker, LC3.

The 3-, 9- and month-old rats in the normal saline groups and the month-old rats in D-galactose group represent the young period, early adult period, late adult period and old period, respectively.

Previous research has suggested that autophagy has an anti-apoptotic effect in injured spinal cord neurons However, the mRNA and protein levels of pro-apoptotic protein, BAX, demonstrated no significant changes at 3 months in the mimetic aging groups, but increased significantly at 9 and 15 months compared with those in the age-matched NS control groups Fig.

The immunofluorescence images demonstrated that BCL2 was increased from 3 to 15 months in the NS group, while it decreased with aging in the D-gal group Fig. Aa-c Reverse transcription-quantitative polymerase chain reaction and Ad-f western blotting were used to study the mRNA and protein expression levels in the D-gal group and NS group, respectively. To investigate the hypotheses regarding the activation and inhibition of autophagy, the protein expression levels of the AMPK-mTOR-ULK1 signaling pathway in the auditory cortex were detected using western blotting.

Representative western blots demonstrated that the expression of p-AMPK was significantly increased in a compensatory manner at 3 months, but significantly decreased at 9 and 15 months in the D-gal groups compared with its expression in the age-matched NS group.

Additionally, the autophagy-related protein Atg13 was significantly increased at 3 and 9 months in the D-gal group compared with the levels in the age-matched NS group Fig.

The 4EBP1 phosphorylation level was also investigated. As demonstrated in Fig. However, the autophagy-related proteins Atg5 and Atg7 were significantly increased at 3 months, while significantly decreased at 15 months in D-gal groups compared with the levels in the age-matched NS group. These results suggest that there is no obvious relevance between 4EBP1 activity and autophagy regulation. Based on these findings, it was hypothesized that p-4EBP1 was not involved in the inhibition of autophagy in the present model.

Changes to the p-4EBP1 protein expression levels in the auditory cortex with aging in the NS and D-gal-induced mimetic aging rats. There was no obvious correlation between p-4EBP1 and autophagy-related proteins. NS, normal saline; D-gal, D-galactose; p, phosphorylated; Atg, autophagy-related protein. Additionally, AMPK may directly activate autophagy through other mechanisms. Autophagy and BCL2 cooperate against apoptosis and delay the process of senescence. Autophagy is a cellular catabolic process that is essential for survival, differentiation, development and homeostasis A large body of evidence indicates that autophagy declines with aging 48 , However, age-related increases of autophagy in rat nucleus pulposus and pancreatic islet cells have also been reported 50 , Recent research has suggested that autophagy increases from the perinatal period to adulthood and then declines after the age of 12 months in the inner ear of mice However, no studies have examined autophagy in the auditory cortex and the changes with age.

In the present study, 3-, 9- and month-old rats in the natural aging and D-gal induced mimetic aging groups were observed. The present results demonstrated that autophagy-related proteins, LC3 and BECN1, were increased from 3 months to 15 months in the natural aging group and decreased at 15 months in the mimetic aging group, while the degradation of p62 demonstrated an inverse trend.

These results suggest that autophagy level is time-dependent and increases from young to adult rats and decreases at an old age in the auditory cortex of SD rats. Ultrastructural morphology of neurons and cell apoptosis level in the mimetic aging group demonstrated no significant differences with those of the age-matched NS group, while the autophagy level increased.

However, at 15 months, it was observed that autophagy in the mimetic aging group was significantly decreased compared to that in the age-matched natural aging group, and neuron degeneration and cell apoptosis were more severe than that in the NS group, as expected.

It was concluded that autophagy serves a protective role in the degeneration of the auditory cortex, and a compensatory increase in autophagy at 3 months protected the auditory cortex from D-gal-induced premature senescence. Deficient or impaired autophagy lost its protective function, leading to the acceleration of apoptosis and finally senescence. The protective role of autophagy was further demonstrated by investigating anti-apoptotic protein levels of BCL2 and BCL-xL in the present study.

Activation of AMPK was significantly increased in the mimetic aging group at 3 months, and p-mTOR and p-ULK1 Ser levels decreased, which could account for the rise of autophagy at 3 months in the mimetic aging group. It was hypothesized that the loss of activation by AMPK and increased inhibition by mTOR failed to suppress autophagy activity until 15 months. Previous research has indicated that increased 4EBP1 activity results in the impairment of autophagy in H 2 O 2 -induced premature senescence in auditory cells However, by detecting 4EBP1 activity and autophagy-related proteins, Atg5 and Atg7, the present study demonstrated that 4EBP1 phosphorylation was increased at 3 and 9 months in the D-gal group compared with that in the age-matched NS group, and no significant difference was observed at 15 months.

Atg5 and Atg7 were increased at 3 months and decreased at 15 months, indicating that 4EBP1 activity may be not involved in the regulation of autophagy in the present model. Additionally, the present results revealed that 4EBP1 phosphorylation may be independent of mTOR, which is consistent with previous studies 25 ,

mTOR: a pharmacologic target for autophagy regulation

Review Free access Phone: Find articles by Kim, Y. Find articles by Guan, K. Published January 2, - More info. Nutrients, growth factors, and cellular energy levels are key determinants of cell growth and proliferation.


Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser and Ser Under nutrient.


The Role of AMPK Activation for Cardioprotection in Doxorubicin-Induced Cardiotoxicity

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mTOR: a pharmacologic target for autophagy regulation

This is an open access article distributed under the terms of Creative Commons Attribution License. Age-related hearing loss ARHL , also known as presbycusis, is a complex degenerative disease characterized by declining auditory function, including increased hearing thresholds and reduced frequency resolution 1. ARHL is one of the most prevalent and chronic conditions of older populations, affecting tens of millions of people worldwide 2 , and may cause social isolation, depression, and even dementia 3. In addition to degeneration of the peripheral auditory organs, auditory cortex degeneration has been demonstrated to serve a crucial role in ARHL pathogenesis 4 , 5 ; however, the molecular mechanism is not well understood. D-galactose D-gal is a reducing sugar and is oxidized into aldehydes and H 2 O 2 when present at high levels 6.

Cardiac troponin I cTnI , and transmission electron microscopy TEM , along with hematoxylin-basic fuchsin-picric acid HBFP staining, were used to evaluate the myocardial ischemic-hypoxic injury and protection. Western blot was used to analyze the relationship of autophagy-associated proteins. Exhaustive exercise caused severe myocardial ischemic-hypoxic injury, which led to an increase in cTnI levels, changes of ischemia—hypoxia, and cells ultrastructure. Compared with the EE group, LEP significantly suppressed exhaustive exercise-induced myocardial injury.

А потом, я не хочу говорить по линии, не защищенной от прослушивания. Глаза Сьюзан расширились. - Как прикажете это понимать. На лице Стратмора тут же появилось виноватое выражение. Он улыбнулся, стараясь ее успокоить.

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

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  • Autophagy is an intracellular degradation pathway targeting organelles and macromolecules, thereby regulating various cellular functions. Erardo P. - 16.05.2021 at 11:28

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