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Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Introduction: A low-vision assessment (LVA) is central to developing a vision rehabilitation plan. However, access to LVAs is often limited by the quantity and geographic distribution of low-vision providers, as well as patient-centred transportation challenges. A tablet-based LVA tool kit, delivered virtually, has the potential to overcome many of these barriers. The purpose of this research was to validate a key component of the tablet-based tool kit - a commercially available iPad visual acuity (VA) test (Eye Chart Pro) iPad app - in a low-vision population., Methods: Participants with low vision ( n  = 26) and those who were normally sighted ( n  = 25) underwent VA testing with both the iPad VA test application and the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. The VA data were compared using a t -test, linear regression and Bland-Altman analysis., Results: There was no significant difference in the mean absolute difference in VA (log of minimum angle of resolution (logMAR)=0.11; p  = 0.82). Eye Chart Pro and Standard ETDRS Chart measures were also not significantly different ( p  = 0.98). However, there were significant differences between test methods in the low-vision group and the normally sighted group ( p  > 0.0001 and p  = 0.007, respectively). The Bland-Altman analysis showed a mean bias (difference) of -0.0005 logMAR between methods, and 95% limits of agreement of 0.298 and -0.299 logMAR., Discussion: The ETDRS chart function on the Eye Chart Pro application can reliably measure VA across a range, from normally sighted patients to those with low vision.

Introduction: A low-vision assessment (LVA) is critical in developing a vision rehabilitation plan. A remotely delivered LVA that replicates a standard in-clinic assessment may bridge the gap for patients not accessing care due to the limited quantity and distribution of low-vision providers. Within an LVA, an accurate and consistent assessment of refraction error is an essential component. No system has currently been validated for the purposes of a remote LVA. The purpose of this study was to validate a commercially available portable refraction approach in a low-vision population., Methods: Low-vision patients ( n  = 26) or normally sighted patients ( n  = 25) underwent a refraction assessment using the Adaptica® 2WIN autorefractor, adaptor scope (Kaleidos) and VisionFit phoropter portable refraction devices, as well as a standard autorefractor (Huvitz) and phoropter (Haag-Streit). Refraction data between systems and populations were compared using intraclass correlations. Bland-Altman plots were used to assess the differences between devices., Results: Spherical equivalent values were found to be reproducible between standard and experimental autorefraction devices (intraclass correlation coefficient (ICC) > 0.8) in both low-vision and normally sighted groups. Similarly, manifest refraction was highly consistent (ICC > 0.8) between devices in all groups. The Bland-Altman plots showed clinically acceptable mean differences of 0.701 between autorefraction methods and -0.116 between manifest refraction methods., Discussion: The 2WIN/VisionFit system can reliably generate refraction values across a spectrum of errors in normally sighted and visually impaired people, and would be feasible to deliver remotely.

Objective  The aim of the study is to compare performance and ease-of-use (EOU) of optic disk assessment using a smartphone direct ophthalmoscope attachment (D-EYE) to the gold standard direct ophthalmoscope (DO). Design  The type of study involved is prospective, randomized, crossover, and educational trial. Participants  The participants involved were first year medical students inexperienced in ophthalmoscopy. Methods  Optic disks of standardized and volunteer patients were examined using the D-EYE and a conventional DO. Optic disk identification, EOU ratings of the devices, self-reported confidence level in their examination with the devices, and estimation of vertical cup-to-disk ratio (VCDR) were compared. Analyses included Chi-square tests, independent samples t -tests, correlations, and multivariable linear regression. Results  Forty-four medical students voluntarily participated in the study. Students using the DO required more attempts (3.57 vs. 2.69, p  = 0.010) and time (197.00 vs. 168.02 seconds, p  = 0.043) to match the patient's fundus to the correct photograph. Overall EOU between the devices (6.40 vs. 4.79, p  < 0.001) and overall confidence in examination (5.65 vs. 4.49, p  = 0.003) were greater when using the D-EYE. There were no statistically significant differences in accuracy of VCDR estimations between the two ophthalmoscopes. Conclusion  Smartphone ophthalmoscopy could offer additional learning opportunities in medical education and may be considered in clinical practice by non-specialist physicians given its greater EOU and increased success in visualizing the optic disk., Competing Interests: Conflict of Interest R.C. reports grants from Physicians' Services Incorporated Grant, during the conduct of the study; D.L. reports grants from Physician's Services Incorporated Grant, during the conduct of the study; I.I. reports grants from Physicians' Services Incorporated Grant, during the conduct of the study; .J.K. reports grants from Physician's Services Incorporated Grant, during the conduct of the study; S.B. reports grants from Physician Services Incorporated, during the conduct of the study; W.H. reports grants from Physician's Services Incorporated Grant, during the conduct of the study; M.X. reports grants from Physicians' Services Incorporated Grant, during the conduct of the study., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. ( https://creativecommons.org/licenses/by-nc-nd/4.0/ ).)

Objective: To characterize the total intraocular aqueous humour antibody profiles in cases receiving anti-vascular endothelial growth factor (anti-VEGF) for retinal vascular disease compared with controls without retinal pathology., Design: Cross-sectional., Participants: 93 aqueous humour samples: 22 eyes undergoing cataract surgery (controls) and 71 eyes receiving intravitreal injections (IVI) (cases) for macular edema or neovascularization., Methods: Antibody isotyping of aqueous humour was performed using Milliplex MAP Human Isotyping Multiplex Assay. Cases and controls were compared for several outcome measures., Results: The primary outcome measure was total mean antibody isotype concentration quantified in the aqueous humour. Secondary outcomes included comparing aqueous humour concentrations with visual acuity, number of IVI received, type of anti-VEGF agent injected, and persistence intra-/subretinal fluid post injection. Mean immunoglobulin M (IgM) concentrations in cases were 19-fold higher compared with controls. Aqueous immunoglobulin G (IgG) 1,2,3,4 and immunoglobulin A (IgA) were 2-4-fold higher in cases compared with controls. Disease-specific trends were observed, with diabetic retinopathy (DR) eyes containing the highest amounts of aqueous antibodies. Total number of injections correlated with higher titres of IgG 1 (p < 0.001), IgG 2 (p < 0.009), and IgG 3 (p < 0.001) in all cases analyzed with the strongest correlations seen in DR eyes (r = 0.77, p < 0.001). Presence of aqueous humour antibodies correlated with worse post-IVI best-corrected visual acuity; IgG 1 (p < 0.01), IgG 2 (p < 0.005), IgG 3 (p < 0.01), and IgA (p < 0.003) in all cases analyzed, with the strongest correlations seen in DR eyes (r = 0.74, p < 0.001)., Conclusions: Intraocular antibodies are present in the aqueous humour at significantly higher concentrations in eyes receiving IVIs for retinal vascular diseases compared with controls., (Copyright © 2019 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD) is a chronic degenerative disease of the retina. Recent reports have highlighted the potential role of mucosal surface microbes in the pathogenesis of AMD. In this case-control study, the composition of the nasal and oral microbiota in newly diagnosed neovascular age-related macular degeneration cases (6 male, 7 female) was compared to controls without retinal diseases (2 male, 3 female). PCR amplification of 16S rRNA genes was performed with universal primers amplifying the V4 variable region (515F-806R). Distinct microbial community characterization was achieved using Principal Coordinates Analysis (PCoA) of the Bray-Curtis index with comparative analysis between cases and controls performed within QIIME 2. Sequencing of all cases and controls revealed clear separation with strong beta diversity between oral and nasal microbial communities (p < 0.001). Microbial composition differed between cases and controls in both oral and nasal samples. The top three oral microbes identified as different compared to controls included Burkholderiales (7.41 log2fold change, p = 3.29E-05), Actinomyceataceae (6.22 log2fold change, p = 3.73E-06) and Gemella (5.28 log2fold change, p = 0.0002). The top three nasal microbes identified as different compared to controls included Rothia (13.6 log2fold change, p =  3.63E-18), Actinobacteria (10.29 log2fold change, p = 9.81E-10) and Propionibacteriales (8.73 log2fold change, p = 6.74E-09). These relative shifts in communities of bacteria detected in newly diagnosed neovascular AMD patients may suggest additional mechanistic links in disease pathogenesis.

Vitamin D has emerged as a potentially important molecule in ophthalmology. To date, all ophthalmic data pertaining to vitamin D has been restricted primarily to tear and serum analysis in human patients. Considering the isolated nature of the eye, we sought to determine the presence of intraocular vitamin D in ocular disease., Methods: 25-Hydroxyvitamin D 3 (25(OH)D 3 ) concentrations were measured in the eye and blood of 120 participants undergoing ophthalmic procedures. Ocular localization of the 1,25-dihydroxyvitamin D 3 -generating (CYP27B1) and deactivating (CYP24A1) hydroxylases was performed by immunohistochemistry. Gene expression of CYP27B1, CYP24A1 and VEGF-A was measured in eyes from patients with and without disease., Results: 25(OH)D 3 was quantified in 112 ocular samples. In 40 cataract patient samples, the average 25(OH)D 3 concentration was 0.057 ng/mL, compared to 72 retinal disease patient samples, average of 0.502 ng/mL (p < 0.001). Intraocular 25(OH)D 3 did not correlate with serum levels of 25(OH)D 3 . There was no difference between the level of 25(OH)D 3 measured in the aqueous and vitreous humour. The vitamin D-specific CYPs 27B1 and 24A1, strongly localized to complementary regions of the ciliary body, retinal pigment epithelium and neural retina. Gene expression analysis confirmed retinal CYP27B1 correlated strongly with VEGF-A in eyes from diabetic patients (r = 0.92, p < 0.001)., Conclusions: Our data confirms that vitamin D is present in the humours of the human eye and that local synthesis/degradation is possible via the ocular CYP27B1 and CYP24A1. This argues for a functional role for local vitamin D production and signaling in the eye and suggests that vitamin D may be an important intraocular mediator in disease pathogenesis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To determine if a narrated white board animation (nWBA) video as part of the consent process for intravenous fluorescein angiography (IVFA) improves patient comprehension compared with a standard consent process., Design: Prospective, randomized study., Participants: Patients undergoing an initial IVFA investigation., Methods: Three groups of 26 patients (N = 78) naïve to the IVFA procedure were included. Groups 1 and 2 consisted of patients undergoing IVFA for diagnostic purposes. Group 1 received the IVFA information via standard physician-patient interaction to obtain standard consent. Group 2 received IVFA information by watching an nWBA explaining the purpose, method, and risks of the diagnostic test to obtain informed consent. Group 3 comprised patients who were not scheduled to undergo IVFA. This group was exposed to both the standard and nWBA consent. All groups completed a 6-question knowledge quiz to assess retained information and a survey to reflect on the consent experience., Results: Participants receiving information via standard physician-patient interaction to obtain informed consent had a lower mean knowledge score (4.38 out of 6; 73%) than participants receiving the information to obtain consent via nWBA (5.04 out of 6, 84%; P = 0.023). Of participants receiving both forms of information (group 3) to obtain informed consent, 73% preferred the nWBA to the standard consent process., Conclusions: Participants receiving consent information for an IVFA diagnostic test via nWBA have better knowledge retention regarding the IVFA procedure and preferred this medium compared with participants receiving the standard physician-patient interaction for obtaining consent. Incorporation of multimedia into the informed consent process should be explored for other diagnostic tests., (Copyright © 2016 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Objective: To examine the tolerability and adverse events reported in the Idiopathic Intracranial Hypertension Treatment Trial (IIHTT)., Methods: Randomized, double-masked, placebo-controlled clinical trial. Trial participants (n = 165) with mild visual loss concurrently receiving low-sodium weight-reduction diet plus the maximally tolerated dosage of acetazolamide (up to 4 g/d) or placebo for 6 months., Main Outcomes Measures: adverse events (AEs), assessment of clinical and laboratory findings at study visits., Results: Thirty-eight of 86 participants randomized to the acetazolamide group (44.1%) tolerated the maximum allowed dosage of 4 g/d. The average time to achieve maximum study dosage in the acetazolamide group was 13 weeks (median 12 weeks; range 10-24 weeks). A total of 676 AEs (acetazolamide, n = 480; placebo, n = 196) and 9 serious AEs (acetazolamide, n = 6; placebo, n = 3) were reported. Notably, the percentages of participants reporting at least 1 AE in the nervous, gastrointestinal, metabolic, and renal organ systems were significantly higher in the acetazolamide group (P < 0.05). The odds of paresthesia (OR 9.82; 95% CI 3.87-27.82), dysgeusia (OR ∞; 95% CI 3.99-∞), vomiting and diarrhea (OR 4.11; 95% CI 1.04-23.41), nausea (OR 2.99; 95% CI 1.26-7.49) and fatigue (OR 16.48; 95% CI 2.39-702.40) were higher in the acetazolamide group than in the placebo group., Conclusion: Acetazolamide appears to have an acceptable safety profile at dosages up to 4 g/d in the treatment of idiopathic intracranial hypertension. The majority of participants in the Idiopathic Intracranial Hypertension Treatment Trial were able to tolerate acetazolamide above 1 g/d for 6 months.

Defects in mitochondrial fission and cyclin dependent kinase 5 (CDK5) activation are early events that precede neuronal loss following NMDA-induced neuronal death. Here, we report that the cytoplasmic CDK5 tightly regulates mitochondrial morphology defects associated with NMDA-induced neuronal injury via regulation of the mitochondrial fission protein, dynamin-related protein 1 (DRP1). We show that DRP1 is a direct target of CDK5. CDK5-mediated phosphorylation of DRP1 at a conserved Serine residue, S585, is elevated at the mitochondria and is associated with increased mitochondrial fission. Ectopic expression of a cytoplasmic CDK5 or mutant DRP1-S585D results in increased mitochondrial fragmentation in primary neurons. Conversely, expression of a dominant negative form of cytoplasmic CDK5 or mutant DRP1-S585A results in elongated mitochondria. In addition, pharmacological inhibition of CDK5 by Roscovitine inhibits DRP1 phosphorylation and mitochondrial fission associated with NMDA-induced neuronal loss. Importantly, conditional deletion of CDK5 significantly attenuates DRP1 phosphorylation at S585 and rescues mitochondrial fission defects in neurons exposed to NMDA. Our studies delineate an important mechanism by which CDK5 regulates mitochondrial morphology defects associated with neuronal injury., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: To investigate the effect of anti-inflammatory therapy on selective laser trabeculoplasty (SLT) outcomes., Design: Randomized, double-masked, placebo-controlled trial., Participants: Patients with primary open-angle or pseudo-exfoliation glaucoma., Methods: Patients undergoing SLT were randomized to receive placebo (artificial tears), prednisolone acetate 1%, or ketorolac tromethamine 0.5% eye drops 4 times per day for 5 days commencing immediately after SLT., Main Outcome Measures: Change in intraocular pressure (IOP) from baseline to the 1-month post-SLT visit., Results: Mean change in IOP at the 1-month primary outcome time point, as well as all other time points, was not significantly different among groups (P = 0.99). Likewise, a repeated-measures, mixed-effects model did not find significant differences in IOP outcome at the 1-month time point (P = 0.95). The IOP was reduced in all groups at the 1-month post-SLT time point and all other time points, and no significant differences were found between groups using separate unadjusted cross-sectional analyses of variance (P > 0.15 for analyses at all time points). Treatment failure rates were not different among groups (P = 0.75), and at 1 year after SLT, the percentage of patients maintaining a 20% IOP reduction ranged from 18% to 22% in the 3 study groups., Conclusions: Anti-inflammatory therapy after SLT does not seem to substantially influence the IOP-lowering effect of SLT. In this study of patients with low baseline IOP, SLT showed limited efficacy in achieving a sustained reduction in IOP., (Copyright © 2014 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.

PGC-1α is an important transcriptional coactivator that plays a key role in mediating mitochondrial biogenesis. Within seconds of the onset of contractile activity, a number of rapid cellular events occur that form part of the initial signaling processes involved in PGC-1α gene regulation, such as elevations in cytoplasmic calcium, AMPK and p38 activation, and elevated ROS production. We observed that basal levels of PGC-1α promoter activity were more sensitive to resting Ca(2+) levels, compared to ROS, p38 or, AMPK signaling. Moreover, enhanced PGC-1α transcription and post-translational activity on DNA were a result of the activation of multiple signal transduction pathways during contractile activity of myotubes. AMPK, ROS, and Ca(2+) appear to be necessary for the regulation of contractile activity-induced PGC-1α gene expression, governed partly through p38 MAPK and CaMKII activity. Whether these signaling pathways are arranged as a linear sequence of events, or as largely independent pathways during contractile activity, remains to be determined., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Objective: Helmet use is the primary form of head protection against traumatic brain injury. Although helmet designs have proven to be effective in reducing the incidence of skull fracture and major traumatic brain injury, there is little evidence that helmets protect against concussion. Linear and rotational accelerations are important mechanisms underlying concussion, yet current testing protocols do not account for rotational acceleration. Technical considerations have prevented a valid, accurate, and reproducible testing paradigm. Our objectives were to design a novel helmet-testing methodology that accurately and reliably measures rotational acceleration at injury-relevant impact forces, locations, and planes and to evaluate differences in rotational force protection in commercially available helmets., Setting: Laboratory study., Intervention: The Kingston Impact Simulator (KIS unit) was used to study 10 commercially available hockey helmets. The rotational acceleration force protection was measured in the horizontal, coronal, and sagittal planes at each of 12 predetermined impact locations., Results: Mean peak unhelmeted and helmeted accelerations at all impact locations and planes ranged from 63 to 28.6 g and from 26.8 to 8.0 g, respectively. The percent reduction in rotational acceleration for all test helmets ranged from 6.4% to 84%. Statistically significant differences in rotational acceleration between manufacturers and within a helmet brand were identified., Conclusions: KIS is a novel testing methodology that identifies rotation force protection within and between hockey helmet models and manufacturers at different impact location and planes. This information may be useful in improving future helmet design and construction to provide maximal protection against the forces causing concussion.

Objective: To evaluate the efficacy of a combination anaesthetic plus dilating gel (ADG) on pupil dilation (PD) and corneal anaesthesia (KA) compared to traditional preoperative pharmacotherapy for cataract surgery., Design: Prospective, noninferiority study., Methods: We studied 20 consenting adults who experienced unilateral cataracts and underwent routine cataract surgery, receiving the traditional preoperative pharmacologic regimen in the operated eye (control eye): diclofenac 0.1%, gentamicin 0.3%, cyclopentolate 1%, phenylephrine 2.5%, and tropicamide 1% 60 and 20 minutes prior to surgery. They then received tetracaine 0.5% and povidone-iodine 5% 10 minutes prior to surgery; and were given tetracaine 0.5%, povidone-iodine 5%, and lidocaine 2% gel 1 minute prior to surgery. Epinephrine 0.1%, 1 cc per 500 mL bag of balanced saline salt solution was administered during surgery. The nonoperated eye (study eye) received tetracaine 0.5%, povidone-iodine 5%, and 0.35 cc ADG gel (phenylephrine 10%, tropicamide 1%, diclofenac 0.1%, and lidocaine 2%) 60 and 10 minutes prior to surgery. PD and KA were measured at baseline, at 30 minutes, and at 5 minutes prior to surgery, and at 5 minutes after surgery., Results: There was no difference in PD (p = 0.2634) or KA (p = 0.6058) between the study eyes and the control eyes at baseline. Preoperatively, greater mydriasis was achieved in the study eye (7.95 ± 0.91 mm vs 7.17 ± 1.25 mm; p < 0.0001). There was no significant difference in preoperative KA between the study and control eyes (1.5 ± 2.2 mm vs 1.4 ± 2.1 mm; p = 0.77)., Conclusions: The combination ADG for preoperative preparation of cataract patients achieves at least equivalent dilation and corneal anaesthesia as the current preoperative pharmacologic regimen., (Copyright © 2012 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H(2)O(2) in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.

The identification of the molecular mechanisms underlying neuronal survival continues to be the subject of intensive research efforts as the incidence of age-related neurodegenerative diseases rises. Amid a complex mélange of environmental and genetic factors that contribute to disease manifestation, much effort has been dedicated to understanding the underlying signaling mechanisms that regulate neuronal survival. A recent study by Yang et al. sheds new light on an intracellular quality-control system that regulates the constitutive abundance of a neuronal survival factor through chaperone-mediated autophagy and links the deregulation of this pathway to Parkinson's disease. Although the primary function of autophagy in most cell types has commonly been thought to be an adaptive response to starvation, it has been proposed that proper functioning of this system is essential for neuronal survival and that its deregulation leads to neurodegeneration.

Reactive oxygen species (ROS) play an important role in cellular function via the activation of signaling cascades. ROS have been shown to affect mitochondrial biogenesis, morphology, and function. Their beneficial effects are likely mediated via the upregulation of transcriptional regulators such as peroxisome proliferator-activated receptor-gamma coactivator-1 protein-alpha (PGC-1alpha). However, the ROS signals that regulate PGC-1alpha transcription in skeletal muscle are not understood. Here we examined the effect of H2O2 on the regulation of PGC-1alpha expression, and its relationship to AMPK activation. We demonstrate that 24 h of exogenous H2O2 treatment increased PGC-1alpha promoter activity and mRNA expression. Both effects were blocked with the addition of N-acetylcysteine, a ROS scavenger. These effects were mediated, in part, via upstream stimulatory factor-1/Ebox DNA binding and involved 1) interactions with downstream sequences and 2) the activation of AMPK. Elevated ROS led to the activation of AMPK, likely via a decline in ATP levels. The activation of AMPK using 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside increased PGC-1alpha promoter activity and mRNA levels but reduced ROS production. Thus the net effect of AMPK activation on PGC-1alpha expression was a result of increased transcriptional activation, counterbalanced by reduced ROS production. The effects of H2O2 on PGC-1alpha expression differed depending on the level of ROS within the cell. Low levels of ROS result in reduced PGC-1alpha mRNA in the absence of an effect on PGC-1alpha promoter activation. In contrast, elevated levels of H2O2 induce PGC-1alpha transcription indirectly, via AMPK activation. These data identify unique interactions between ROS and AMPK activation on the expression of PGC-1alpha in muscle cells.

Thyroid hormone (T(3)) regulates the function of many tissues within the body. The effects of T(3) have largely been attributed to the modulation of thyroid hormone receptor-dependent gene transcription. However, nongenomic actions of T(3) via the initiation of signaling events are emerging in a number of cell types. This study investigated the ability of short-term T(3) treatment to phosphorylate and, therefore, activate signaling proteins in rat tissues in vivo. The kinases investigated included p38, AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK) 1/2. Following 2 h of T(3) treatment, p38 and AMPK phosphorylation was increased in both the slow-twitch soleus and the fast-twitch plantaris muscles. In contrast, ERK1/2 was not activated in either muscle type. Neither p38 nor AMPK was affected in heart. However, AMPK activation was decreased by T(3) in liver. ERK1/2 activation was decreased by T(3) in heart, but increased in liver. Possible downstream consequences of T(3)-induced kinase phosphorylation were investigated by measuring cAMP response element binding protein (CREB) and thyroid hormone receptor DNA binding, as well as peroxisome proliferator-activated receptor-alpha coactivator-1 mRNA levels. Protein DNA binding to the cAMP or thyroid hormone response elements was unaltered by T(3). However, peroxisome proliferator-activated receptor-alpha coactivator-1 mRNA expression was increased following 12 h of T(3) treatment in soleus. These data are the first to characterize the effects of T(3) treatment on kinase phosphorylation in vivo. We show that T(3) rapidly modifies kinase activity in a tissue-specific fashion. Moreover, the T(3)-induced phosphorylation of p38 and AMPK in both slow- and fast-twitch skeletal muscles suggests that these events may be important in mediating hormone-induced increases in mitochondrial biogenesis in skeletal muscle.

The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

Skeletal muscle is a highly malleable tissue, capable of pronounced metabolic and morphological adaptations in response to contractile activity (i.e. exercise). Each bout of contractile activity results in a coordinated alteration in the expression of a variety of nuclear DNA and mitochondrial DNA (mtDNA) gene products, leading to phenotypic adaptations. This results in an increase in muscle mitochondrial volume and changes in organelle composition, referred to as mitochondrial biogenesis. The functional consequence of this biogenesis is an improved resistance to fatigue. Signals initiated by the exercise bout involve changes in intracellular Ca2+ as well as alterations in energy status (i.e. ATP/ADP ratio) and the consequent activation of downstream kinases such as AMP kinase and Ca2+-calmodulin-activated kinases. These kinases activate transcription factors that bind DNA to affect the transcription of genes, the most evident manifestation of which occurs during the post-exercise recovery period when energy metabolism is directed toward anabolism, rather than contractile activity. An important protein that is affected by exercise is the transcriptional coactivator PGC-1alpha, which cooperates with multiple transcription factors to induce the expression of nuclear genes encoding mitochondrial proteins. Once translated in the cytosol, these mitochondrially destined proteins are imported into the mitochondrial outer membrane, inner membrane or matrix space via specific import machinery transport components. Contractile activity affects the expression of the import machinery, as well as the kinetics of import, thus facilitating the entry of newly synthesized proteins into the expanding organelle. An important set of proteins that are imported are the mtDNA transcription factors, which influence the expression and replication of mtDNA. While mtDNA contributes only 13 proteins to the synthesis of the organelle, these proteins are vital for the proper assembly of multi-subunit complexes of the respiratory chain, when combined with nuclear-encoded protein subunits. The expansion of skeletal muscle mitochondria during organelle biogenesis involves the assembly of an interconnected network system (i.e. a mitochondrial reticulum). This expansion of membrane size is influenced by the balance between mitochondrial fusion and fission. Thus, mitochondrial biogenesis is an adaptive process that requires the coordination of multiple cellular events, including the transcription of two genomes, the synthesis of lipids and proteins and the stoichiometric assembly of multisubunit protein complexes into a functional respiratory chain. Impairments at any step can lead to defective electron transport, a subsequent failure of ATP production and an inability to maintain energy homeostasis.

The early cellular signals associated with contractile activity initiate the activation and induction of transcription factors that regulate changes in skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum response factor (SRF) are potentially important mediators of mitochondrial biogenesis based on the prevalence of binding sites for them in the promoter regions of genes encoding mitochondrial proteins, including PGC-1 alpha, the important regulator of mitochondrial biogenesis. Thus, to further define a role for transcription factors at the onset of contractile activity, we examined the time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in electrically stimulated mouse C(2)C(12) skeletal muscle cells. Early transient increases in Egr-1 mRNA levels within 30 min (P < 0.05) of contractile activity led to threefold increases (P < 0.05) in Egr-1 protein by 60 min. The increase in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life after 30 min of stimulation showed only a 58% decline. Stimulation of muscle cells had no effect on Sp1 mRNA but led to progressive increases (P < 0.05) in SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but occurred coincident with increases (P < 0.05) in SRF-serum response element DNA binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To assess the importance of the recovery period, 12 h of continuous contractile activity was compared with four successive 3-h bouts, with an intervening 21-h recovery period after each bout. Continuous contractile activity led to a twofold increase (P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43% decrease in Sp1 mRNA expression. The recovery period prevented the decline in Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus continuous and intermittent contractile activity evoked different specific transcription factor expression patterns, which may ultimately contribute to divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle.

Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca(2+) in muscle cells. To characterize the potential role of Ca(2+) as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca(2+) ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 microM A-23187 for 48-96 h increased nuclear-encoded beta-subunit F(1)ATPase and malate dehydrogenase (MDH) mRNA levels by 50-100% (P < 0.05) but decreased mRNA levels of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially encoded subunits COX II and COX III were decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca(2+) differentially regulates the mRNA level of nuclear and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam levels by 48 h. A-23187 induced time-dependent increases in Egr-1 mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also increased Egr-1, serum response factor, and Sp1 protein expression, transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or beta-subunit F(1)ATPase mRNA. These results indicate that changes in intracellular Ca(2+) can modify mitochondrial phenotype, in part via the involvement of Egr-1.

The transcriptional coactivator the peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been identified as an important mediator of mitochondrial biogenesis based on its ability to interact with transcription factors that activate nuclear genes encoding mitochondrial proteins. The induction of PGC-1alpha protein expression under conditions that provoke mitochondrial biogenesis, such as contractile activity or thyroid hormone (T(3)) treatment, is not fully characterized. Thus we related PGC-1alpha protein expression to cytochrome c oxidase (COX) activity in 1) tissues of varying oxidative capacities, 2) tissues from animals treated with T(3), and 3) skeletal muscle subject to contractile activity both in cell culture and in vivo. Our results demonstrate a strong positive correlation (r = 0.74; P < 0.05) between changes in PGC-1alpha and COX activity, used as an index of mitochondrial adaptations. The highest constitutive levels of PGC-1alpha were found in the heart, whereas the lowest were measured in fast-twitch white muscle and liver. T(3) increased PGC-1alpha content similarly in both fast- and slow-twitch muscle, as well as in the liver, but not in heart. T(3) also induced early (6 h) increases in AMP-activated protein kinase (AMPKalpha) activity, as well as later (5 day) increases in p38 MAP kinase activity in slow-twitch, but not in fast-twitch, muscle. Contractile activity provoked early increases in PGC-1alpha, coincident with increases in mitochondrial transcription factor A (Tfam), and nuclear respiratory factor-1 (NRF-1) protein expression, suggesting that PGC-1alpha is physiologically important in coordinating the expression of the nuclear and mitochondrial genomes. Ca(2+) ionophore treatment of muscle cells led to an approximately threefold increase in PGC-1alpha protein, and contractile activity induced rapid and marked increases in both p38 MAP kinase and AMPKalpha activities. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) treatment of muscle cells also led to parallel increases in AMPKalpha activity and PGC-1alpha protein levels. These data are consistent with observations that indicate that increases in PGC-1alpha protein are affected by Ca(2+) signaling mechanisms, AMPKalpha activity, as well as posttranslational phosphorylation events that increase PGC-1alpha protein stability. Our data support a role for PGC-1alpha in the physiological regulation of mitochondrial content in a variety of tissues and suggest that increases in PGC-1alpha expression form part of a unifying pathway that promotes both T(3)- and contractile activity-induced mitochondrial adaptations.

Behavioural and hereditary conditions are known to decrease mitochondrial volume and function within skeletal muscle. This reduces endurance performance, and is manifest both at high- and low-intensity levels of exertion. A programme of regular endurance exercise, undertaken over a number of weeks, produces significant adaptations within skeletal muscle such that noticeable improvements in oxidative capacity are evident, and the related decline in endurance performance can be attenuated. Notwithstanding the important implications that this has for the highly trained endurance athlete, an improvement in mitochondrial volume and function through regular physical activity also endows the previously sedentary and/or aging population with an improved quality of life, and a greater functional independence. An understanding of the molecular and cellular mechanisms that govern the increases in mitochondrial volume with repeated bouts of exercise can provide insights into possible therapeutic interventions to care for those with mitochondrially-based diseases, and those unable to withstand regular physical activity. This review focuses on the recent developments in the molecular aspects of mitochondrial biogenesis in chronically exercising muscle. Specifically, we discuss the initial signalling events triggered by muscle contraction, the activation of transcription factors involved in both nuclear and mitochondrial DNA transcription, as well as the post-translational import mechanisms required for mitochondrial biogenesis. We consider the importance and relevance of chronic physical activity in the induction of mitochondrial biogenesis, with particular emphasis on how an endurance training programme could positively affect the age-related decline in mitochondrial content and delay the progression of age- and physical inactivity-related diseases.

Regularly performed exercise in the form of endurance training produces a well-established adaptation in skeletal muscle termed mitochondrial biogenesis. The physiological benefit of this is an enhanced performance of muscle when subject to endurance exercise. This is not only of great advantage for athletic endeavours, but it also clearly improves the quality of life of previously sedentary individuals and those involved in injury rehabilitation. Here we review the molecular basis for mitochondrial biogenesis in muscle, from the initial signals arising in contracting muscle, to the transcription factors involved in mitochondrial and nuclear DNA transcription, as well as the post-translational import mechanisms required for the synthesis of the organelle. We discuss specific protein components associated with reactive oxygen species production, and suggest some questions which remain unanswered with respect to the role of exercise-induced mitochondrial biogenesis in ageing, apoptosis and disease.

Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (-726 base pairs) or minimal (-66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the -726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na(+) channel blocker tetrodotoxin, the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (+75 to +104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.