Effect of statin on age-related hearing loss via drug repurposing

Hearing loss in the elderly cause communication difficulties, decreased quality of life, isolation, loneliness and frustration. The aim of our study was to investigate the effect of drug repurposing candidates in aging mouse. The selected candidate drugs for age-related hearing loss (ARHL) included atorvastatin (AS) and sarpogrelate. Monotherapy or fixed dose combination (FDC) products were administered via oral gavage for 6 consecutive months. Auditory outcomes showed significant hearing preservation in AS-treated aging mice compared to aging control, especially in the early stages of ARHL in both 8 and 16 kHz frequencies. However, none of the FDC products were able to prevent ARHL regardless of AS involvement. In aging mice, damage and dysfunction of mitochondria was noted as well as reactive oxygen species overproduction leading to oxidative stress and intrinsic apoptosis. These processes of ARHL were significantly prevented with administration of AS. Normal structures of mitochondria were maintained, and antioxidant activity were proceeded by activation of HSF1/Sirt1 pathway. Our study suggests that AS is a promising drug repurposing candidate to delay the progression of ARHL.     

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age‐related hearing impairment

Age‐related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD⁺/NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP‐1, and PGC‐1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD⁺ levels on aging and age‐related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of β‐lapachone (β‐lap), a known plant‐derived metabolite that modulates cellular NAD⁺ by conversion of NADH to NAD⁺ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD⁺ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro‐inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF‐κB, p53, and IDH2. However, augmentation of NAD⁺ by β‐lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD⁺ levels by pharmacological agents may be a tangible therapeutic option for treating various age‐related diseases, including ARHL.     

Identifying MicroRNAs Involved in Degeneration of the Organ of Corti during Age-Related Hearing Loss

MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.     

The expression of PHB2 in the cochlea: Possible relation to age-related hearing loss

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI-OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI-OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H₂O₂ treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment.     

Degradation of cochlear Connexin26 accelerate the development of age-related hearing loss

The GJB2 gene, encoding Connexin26 (Cx26), is one of the most common causes of inherited deafness. Clinically, mutations in GJB2 cause congenital deafness or late-onset progressive hearing loss. Recently, it has been reported that Cx26 haploid deficiency accelerates the development of age-related hearing loss (ARHL). However, the roles of cochlear Cx26 in the hearing function of aged animals remain unclear. In this study, we revealed that the Cx26 expression was significantly reduced in the cochleae of aged mice, and further explored the underlying molecular mechanism for Cx26 degradation. Immunofluorescence co-localization results showed that Cx26 was internalized and degraded by lysosomes, which might be one of the important ways for Cx26 degradation in the cochlea of aged mice. Currently, whether the degradation of Cx26 in the cochlea leads directly to ARHL, as well as the mechanism of Cx26 degradation-related hearing loss are still unclear. To address these questions, we generated mice with Cx26 knockout in the adult cochlea as a model for the natural degradation of Cx26. Auditory brainstem response (ABR) results showed that Cx26 knockout mice exhibited high-frequency hearing loss, which gradually progressed over time. Pathological examination also revealed the degeneration of hair cells and spiral ganglions, which is similar to the phenotype of ARHL. In summary, our findings suggest that degradation of Cx26 in the cochlea accelerates the occurrence of ARHL, which may be a novel mechanism of ARHL.     

Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.     

Grifola frondosa (Maitake) extract activates PPARδ and improves glucose intolerance in high-fat diet-induced obese mice

Grifola frondosa is a mushroom that has anti-obesity effects, but the detailed mechanism is poorly understood. In this study, we found that lipid soluble extracts derived from G. frondosa (GFE) had peroxisome proliferator-activated receptor δ (PPARδ) agonist activity, inducing the expression of PPARδ-target genes in vitro. Furthermore, administration of GFE to high-fat diet-induced obese mice lowered the total blood cholesterol levels, upregulated the expression of PPARδ-target genes in skeletal muscles and improved glucose intolerance. Additionally, analyses of C₂C₁₂ myotubes revealed that GFE restored glucose uptake, which was inhibited by sodium palmitate, to normal levels. Unexpectedly, such acceleration was not abolished by a PPARδ antagonist. These results suggest that G. frondosa is a novel functional food that may prevent life-style related diseases like obesity and diabetes, and that these beneficial effects are likely to be mediated through the activation of PPARδ and a PPARδ-independent insulin signaling pathway.     

Long-Term Bezafibrate Treatment Improves Skin and Spleen Phenotypes of the mtDNA Mutator Mouse

Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent's liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.     

NAD+ supplementation rejuvenates aged gut adult stem cells

The tissue decline due to aging is associated with the deterioration of adult stem cell function. Here we show the number and proliferative activity of intestinal stem cells (ISCs) but not Paneth cells decline during aging, as does ISC function assessed ex vivo. Levels of SIRT1 and activity of mTORC1 also decline with aging. The treatment with the NAD(+) precursor nicotinamide riboside (NR) rejuvenates ISCs from aged mice and reverses an impaired ability to repair gut damage. The effect of NR is blocked by the mTORC1 inhibitor rapamycin or the SIRT1 inhibitor EX527. These findings demonstrate that small molecules affecting the NAD/SIRT1/mTORC1 axis may guide a translational path for maintenance of the intestine during aging.     

The metabolic syndrome and the hepatic fatty acid drainage hypothesis

Much data indicates that lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver metabolism towards activation of peroxisome proliferator activated receptor (PPAR)alpha-regulated fatty acid catabolism in mitochondria. Feeding rats with lipid lowering agents leads to hypolipidemia, possibly by increased channeling of fatty acids to mitochondrial fatty acid oxidation at the expense of triglyceride synthesis. Our hypothesis is that increased hepatic fatty acid oxidation and ketogenesis drain fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects on fat mass accumulation and improved peripheral insulin sensitivity. To investigate this theory we employ modified fatty acids that change the plasma profile from atherogenic to cardioprotective. One of these novel agents, tetradecylthioacetic acid (TTA), is of particular interest due to its beneficial effects on lipid transport and utilization. These hypolipidemic effects are associated with increased fatty acid oxidation and altered energy state parameters of the liver. Experiments in PPAR alpha-null mice have demonstrated that the effects hypolipidemic of TTA cannot be explained by altered PPAR alpha regulation alone. TTA also activates the other PPARs (e.g., PPAR delta) and this might compensate for deficiency of PPAR alpha. Altogether, TTA-mediated clearance of blood triglycerides may result from a lowered level of apo C-III, with a subsequently induction of hepatic lipoprotein lipase activity and (re)uptake of fatty acids from very low density lipoprotein (VLDL). This is associated with an increased hepatic capacity for fatty acid oxidation, causing drainage of fatty acids from the blood stream. This can ultimately be linked to hypolipidemia, anti-adiposity, and improved insulin sensitivity.     

Generation,Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other''s NAD supply by providing alternative precursors.     

Phosphoinositide kinase signaling controls ER-PM cross-talk

Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca²⁺-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions.     

Apolipoprotein D overexpression alters hepatic prostaglandin and omega fatty acid metabolism during the development of a non-inflammatory hepatic steatosis

Apolipoprotein D (ApoD) is a secreted lipocalin associated with neuroprotection and lipid metabolism. Overexpression of ApoD in mouse neural tissue induces the development of a non-inflammatory hepatic steatosis in 12-month-old transgenic animals. Previous data indicates that accumulation of arachidonic acid, ApoD's preferential ligand, and overactivation of PPARγ are likely the driving forces in the development of the pathology. However, the lack of inflammation under those conditions is surprising. Hence, we further investigated the apparent repression of inflammation during hepatic steatosis development in aging transgenic animals. The earliest modulation of lipid metabolism and inflammation occurred at 6 months with a transient overexpression of L-PGDS and concomitant overproduction of 15d-PGJ₂, a PPARγ agonist. Hepatic lipid accumulation was detectable as soon as 9 months. Inflammatory polarization balance varied in time, with a robust anti-inflammatory profile at 6 months coinciding with 15d-PGJ₂ overproduction. Omega-3 and omega-6 fatty acids were preferentially stored in the liver of 12-month-old transgenic mice and resulted in a higher omega-3/omega-6 ratio compared to wild type mice of the same age. Thus, inflammation seems to be controlled by several mechanisms in the liver of transgenic mice: first by an increase in 15d-PGJ₂ production and later by a beneficial omega-3/omega-6 ratio. PPARγ seems to play important roles in these processes. The accumulation of several omega fatty acids species in the transgenic mouse liver suggests that ApoD might bind to a broader range of fatty acids than previously thought.     

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging‐related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro‐apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.