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Extracellular vesicles (EVs) are small packages that are released by almost all types of cells. While the role of EVs in pathogenesis of certain diseases such as cancer is well established, EVs role in ocular health and disease is still at early stages of investigation. Given the significant role of EVs in pathological development and progression of diseases such as cancer, EVs present a similar opportunity for investigation in ocular pathophysiology. Studies have shown the presence of EVs in fluids from the ocular environment have close links with ocular health and disease. Hence, the cargo carried in EVs from ocular fluids can be used for monitoring disease phenotypes or therapeutic outcomes in eye‐related disorders. Furthermore, in recent times EVs have increasingly gained attention as therapeutics and drug‐delivery vehicles for treatment of eye diseases. There is a close relationship between EVs and mitochondria functioning with mitochondria dysfunction leading to a significant number of ophthalmic disorders. This review discusses the current knowledge of EVs in visual systems with a special focus on eye diseases resulting from dysfunctional mitochondria. [ABSTRACT FROM AUTHOR]

Abstract: Background: Diabetes is a metabolic syndrome that results in chronically increased blood glucose (hyperglycaemia) due to defects either in insulin secretion consequent to the loss of beta cells in the pancreas (type 1) or to loss of insulin sensitivity in target organs in the presence of normal insulin secretion (type 2). Long term hyperglycaemia can lead to a number of serious health-threatening pathologies, or complications, especially in the kidney, heart, retina and peripheral nervous system. Scope of review: Here we summarise the current literature on the role of the mitochondria in complications associated with diabetes, and the limitations and potential of rodent models to explore new modalities to limit complication severity. Major conclusions: Prolonged hyperglycaemia results in perturbation of catabolic pathways and in an over-production of ROS by the mitochondria, which in turn may play a role in the development of diabetic complications. Furthermore, current models don't offer a comprehensive recapitulation of these complications. General significance: The onset of complications associated with type 1 diabetes can be varied, even with tightly controlled blood glucose levels. The potential role of inherited, mild mitochondrial dysfunction in accelerating diabetic complications, both in type 1 and 2 diabetes, remains unexplored. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. [Copyright &y& Elsevier]

Abstract: Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II+III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II+III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways. [Copyright &y& Elsevier]

With the advancement of various gene transfer technologies, the establishment of mitochondria transfer as a viable technique to genetically engineer mouse models paradoxically lagged behind other genetic technologies. The lack of demonstrable recombination in mtDNA necessitates different approaches to conventional transgenesis-based techniques. Initially, heteroplasmic mice were created to explore disease pathogenesis and mitochondrial dynamics in an in vivo system. Ultimately, transmitochondrial mouse models will be used to explore the role of the mitochondrial genome in human disease processes and in the development of novel human gene therapies. Here, we describe methodology to produce transmitochondrial mice (both homoplasmic and heteroplasmic models) harboring foreign mitochondrial genomes, using both embryo microinjection and embryonic stem (ES) cell-based approaches. Specific modeling and the procedures for mitochondrial transfer will be of considerable importance toward our understanding of discrete mitochondrial mutations, as well as lead to the development of novel strategies and therapies for human diseases influenced by mitochondrial DNA mutations. [Copyright &y& Elsevier]

The article discusses treatment of glaucoma. It states as per the study of scientist Williams, P.A., age and declining of nicotinamide adenine dinucleotide (NAD+) identified as a major risk factor for glaucoma. It informs Nmnat1 gene therapy found out to be alternative to NAD+ supplements and good tolerability in the effective treatment of glaucoma.

Despite clear evidence of a neuroprotective physiological role of amyloid precursor protein (APP) and its non-amyloidogenic processing products, APP has been investigated mainly in animal and cellular models of amyloid pathology in the context of Alzheimer's disease. The rare familial mutations in APP and presenilin-1/2, which sometimes drive increased amyloid β (Aβ) production, may have unduly influenced Alzheimer's disease research. APP and its cleavage products play important roles in cellular and mitochondrial metabolism, but many studies focus solely on Aβ. Mitochondrial bioenergetic metabolism is essential for neuronal function, maintenance and survival, and multiple reports indicate mitochondrial abnormalities in patients with Alzheimer's disease. In this review, we focus on mitochondrial abnormalities reported in sporadic Alzheimer's disease patients and the role of full-length APP and its non-amyloidogenic fragments, particularly soluble APPα, on mitochondrial bioenergetic metabolism. We do not review the plethora of animal and in vitro studies using mutant APP/presenilin constructs or experiments using exogenous Aβ. In doing so, we aim to invigorate research and discussion around non-amyloidogenic APP processing products and the mechanisms linking mitochondria and complex neurodegenerative disorders such as sporadic Alzheimer's disease. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc. [ABSTRACT FROM AUTHOR]

The article provides information about hereditary eye diseases and the hidden abnormalities that go with it. It is mentioned that autosomal dominant optic atrophy (ADOA) is a subtle chronic type of the sudden visual loss typical of Leber hereditary optic neuropathy (LHON). It is also mentioned that the spectrum of extraocular neurological features described in patients with LHON were also observed in individuals with ADOA. A discussion of the histochemical studies conducted by P. Yu-Wai-Man et al is provided.

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death. [ABSTRACT FROM AUTHOR]

We describe a model of acute intraocular pressure (IOP) elevation in the mouse eye that induces reversible loss of inner retinal function associated with oxidative stress, glial cell activation and minimal loss of retinal ganglion cell (RGC) number. Young healthy mouse eyes recover inner retinal function within 7-days but more persistent functional loss is seen in older mice. Manipulation of diet and exercise further modify RGC recovery demonstrating the utility of this injury model for investigating lifestyle and therapeutic interventions. We believe that systematic investigation into the characteristics and determinants of RGC recovery following an IOP challenge will shed light on processes that govern RGC vulnerability in the early stages of glaucoma. [ABSTRACT FROM AUTHOR]

Abstract: We have previously shown that the optic nerve of mice becomes increasingly vulnerable to injury with advancing age. Here, we investigated whether regular exercise can modify this age-related vulnerability and improve optic nerve recovery after injury. Aged (12-month-old) C57BL/6J mice were exercised by swimming for 60 min/d, 5 d/wk for 6 weeks. After 5 weeks, injury to the optic nerve was induced by short-term elevation of intraocular pressure. Retinal function was recorded using the electroretinogram and the cellular and biochemical changes induced by injury were assessed using immunohistochemistry and quantitative polymerase chain reaction. We found that exercise almost completely reversed age-related vulnerability of the optic nerve to injury such that exercised aged mice had a similar functional response to injury as non-exercised young (3-month-old) mice. Exercise also abrogated injury-induced astrocytic gliosis and macrophage activation in the aged retina. These data suggest that the known benefits of exercise also extend to the visual system and support further investigation of physical activity as a means of protecting against injury, dysfunction, and degeneration in the aging eye. [Copyright &y& Elsevier]

Mitochondrial dysfunction increases reactive oxygen species (ROS) production and when this overwhelms the cellular antioxidant defences, oxidative stress ensues. Oxidative stress is recognized as a common pathologic pathway in many neurodegenerative diseases. Recent reports have also demonstrated oxidative stress in ocular tissues derived from experimental glaucoma models and clinical samples. There is also accumulating evidence pointing to mitochondrial dysfunction being present in some glaucoma patients. Thus oxidative stress from mitochondrial dysfunction may also play a causal role in glaucoma. The mechanisms by which oxidative stress may induce retinal ganglion cell loss in glaucoma are not fully understood but could include direct neurotoxic effects from ROS or indirect damage from oxidative stress-induced dysfunction of glial cells. This review will consider the evidence for the presence of oxidative stress in glaucoma; the mechanisms by which oxidative stress may contribute to disease pathogenesis; and also consider therapeutic approaches that target oxidative stress as a means of protecting against optic nerve degeneration. [ABSTRACT FROM AUTHOR]

Summary [ABSTRACT FROM AUTHOR]

Summary: Epilepsy is characterised by recurrent seizures, which are manifestations of aberrant cortical neuronal firing. It is unclear whether oxidative stress is a cause or consequence of seizure-related hippocampal neuronal loss or whether it occurs concomitantly with the initiation of cell death pathways. We utilised the rapid electrical amygdala kindling (REAK) model which does not induce cell death to examine early seizure-induced oxidative stress in wildtype and superoxide dismutase 2 (Sod2) +/− mice, which lack 50% of Sod2 activity and are therefore known to be more susceptible to mitochondrial oxidative stress. A significant increase in lipid peroxidation and superoxide production was noted in the hippocampi of wildtype mice and a more delayed response observed in Sod2 +/− mice at early time-points post-seizures, but protein carbonylation levels appeared unchanged. A 10-fold increase in superoxide production was seen in the Sod2 +/− CA2 neurons, indicating that Sod2 plays an important role in protecting the CA2 region of the hippocampus from seizure-induced free radical damage. Early hippocampal cell death was undetectable in wildtype or Sod2 +/− mice post-seizures. We were able to demonstrate that hippocampal oxidative stress occurred as a direct consequence of seizures rather than downstream of activation of cell death pathways. We were also able to show that this increase in oxidative stress was not sufficient to cause cell death within the time window investigated. Our data indicates that a possible upregulation of endogenous antioxidant activity might exist within selective hippocampal sectors in the Sod2 +/− mice that are as yet unknown. [Copyright &y& Elsevier]

The characterization of mitochondrial diseases has proceeded rapidly since the first descriptions of mitochondrial DNA (mtDNA)-linked disease mutations appeared in the late 1980s. To elucidate mechanisms of a variety of mitochondrial disorders and disease, both in vitro and in vivo modeling systems have been exploited. To produce these models, numerous approaches have been undertaken due to the difficulty associated with targeted mutagenesis and directed modification of the mitochondrial genome. Currently available models of mitochondrial disease are discussed in this paper, including our xenomitochondrial mice. In this model, mitochondria from one donor species are transferred to another. By doing so, cells and animals were generated with varying levels of heteroplasmy (or homoplasmy) for the introduced mitochondrial genomes. This caused graded variations in electron transport chain (ETC) dysfunction which were dependent upon the evolutionary divergence between donor and recipient. The protocol in generating these models involved the utilization of rhodamine-6G (R6G) to remove or eliminate endogenous mtDNA from the recipient cells. This paper will highlight the process and the implications of R6G treatment of mouse embryonic stem (ES) cells to create transmitochondrial cybrids. We summarize the history and mechanism of action of R6G as well as the future prospects for xenomitochondrial models toward increasing our understanding of mitochondrial biology and the dynamic interplay in signaling between mitochondria and the nucleus. [ABSTRACT FROM AUTHOR]

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics. Author summary: Recent studies have highlighted the importance of mitochondrial DNA variation in both primary mitochondrial disease and complex, human pathology including COVID-19, and space-flight stress. The vast amount of existing, next-generation sequencing (NGS) data can be leveraged to interrogate both nuclear and mitochondrial DNA (mtDNA) sequence simultaneously, allowing for analysis of the interplay between mitochondrial and nuclear encoded genes in mitochondrial function. Identifying mtDNA sequence accurately is complicated by the presence of nuclear encoded mitochondrial sequences (NUMTs), which are homologous to mtDNA. Current software for analyzing mtDNA from NGS do not accurately model the unique characteristics of mitochondrial genetics. We introduce MitoScape, a novel, big-data, software which models mitochondrial genetics through machine learning to accurately identify mtDNA sequence from NGS data. MitoScape takes advantage of rho-zero cell data to model the characteristics of NUMTs. We show that MitoScape produces more accurate heteroplasmy estimates compared to published software. We provide an example of applying MitoScape in replicating an association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men. MitoScape is an important contribution to mitochondrial genomics allowing for accurate mtDNA variants, and the ability to tailor mtDNA analysis in different population and disease contexts, which is not available in other software. [ABSTRACT FROM AUTHOR]

Mitochondrial OXPHOS generates most of the energy required for cellular function. OXPHOS biogenesis requires the coordinated expression of the nuclear and mitochondrial genomes. This represents a unique challenge that highlights the importance of nuclear-mitochondrial genetic communication to cellular function. Here we investigated the transcriptomic and functional consequences of nuclear-mitochondrial genetic divergence in vitro and in vivo. We utilized xenomitochondrial cybrid cell lines containing nuclear DNA from the common laboratory mouse Mus musculus domesticus and mitochondrial DNA (mtDNA) from Mus musculus domesticus, or exogenous mtDNA from progressively divergent mouse species Mus spretus, Mus terricolor, Mus caroli and Mus pahari. These cybrids model a wide range of nuclear-mitochondrial genetic divergence that cannot be achieved with other research models. Furthermore, we used a xenomitochondrial mouse model generated in our laboratory that harbors wild-type, C57BL/6J Mus musculus domesticus nuclear DNA and homoplasmic mtDNA from Mus terricolor. RNA sequencing analysis of xenomitochondrial cybrids revealed an activation of interferon signaling pathways even in the absence of OXPHOS dysfunction or immune challenge. In contrast, xenomitochondrial mice displayed lower baseline interferon gene expression and an impairment in the interferon-dependent innate immune response upon immune challenge with herpes simplex virus, which resulted in decreased viral control. Our work demonstrates that nuclear-mitochondrial genetic divergence caused by the introduction of exogenous mtDNA can modulate the interferon immune response both in vitro and in vivo, even when OXPHOS function is not compromised. This work may lead to future insights into the role of mitochondrial genetic variation and the immune function in humans, as patients affected by mitochondrial disease are known to be more susceptible to immune challenges. [ABSTRACT FROM AUTHOR]

• OXPHOS bioenergetic compensation in LHON cells does not explain disease phenotype. • LHON affected cells display transcriptional downregulation of metabolic pathways. • LHON affected cells suffer a growth impairment under metabolic challenge. • LHON unaffected carrier cells are not affected by metabolic challenge. • Decreased susceptibility to metabolic challenge may preserve metabolic homeostasis. Leber hereditary optic neuropathy (LHON) is one of the most common primary mitochondrial diseases. It is caused by point mutations in mitochondrial DNA (mtDNA) genes and in some cases, it can result in irreversible vision loss, primarily in young men. It is currently unknown why LHON mutations affect only some carriers and whether bioenergetic compensation enables unaffected carriers to overcome mitochondrial impairment and preserve cellular function. Here, we conducted bioenergetic metabolic assays and RNA sequencing to address this question using male-only, age-matched, m.11778G > A lymphoblasts and primary fibroblasts from both unaffected carriers and affected individuals. Our work indicates that OXPHOS bioenergetic compensation in LHON peripheral cells does not explain disease phenotype. We show that complex I impairment is similar in cells from unaffected carrier and affected patients, despite a transcriptional downregulation of metabolic pathways including glycolysis in affected cells relative to carriers detected by RNA sequencing. Although we did not detect OXPHOS bioenergetic compensation in carrier cells under basal conditions, our results indicate that cells from affected patients suffer a growth impairment under metabolic challenge compared to carrier cells, which were unaffected by metabolic challenge. If recapitulated in retinal ganglion cells, decreased susceptibility to metabolic challenge in unaffected carriers may help preserve metabolic homeostasis in the face of the mitochondrial complex I bioenergetic defect. [ABSTRACT FROM AUTHOR]

Importance Retinal ganglion cells endure significant metabolic stress in glaucoma but maintain capacity to recover function. Nicotinamide, a precursor of NAD+, is low in serum of glaucoma patients and its supplementation provides robust protection of retinal ganglion cells in preclinical models. However, the potential of nicotinamide in human glaucoma is unknown. Background: To examine the effects of nicotinamide on inner retinal function in glaucoma, in participants receiving concurrent glaucoma therapy. Design Crossover, double‐masked, randomized clinical trial. Participants recruited from two tertiary care centres. Participants: Fifty‐seven participants, diagnosed and treated for glaucoma. Methods: Participants received oral placebo or nicotinamide and reviewed six‐weekly. Participants commenced 6 weeks of 1.5 g/day then 6 weeks of 3.0 g/day followed by crossover without washout. Visual function measured using electroretinography and perimetry. Main outcome measures: Change in inner retinal function, determined by photopic negative response (PhNR) parameters: saturated PhNR amplitude (Vmax), ratio of PhNR/b‐wave amplitude (Vmax ratio). Results: PhNR Vmax improved beyond 95% coefficient of repeatability in 23% of participants following nicotinamide vs 9% on placebo. Overall, Vmax improved by 14.8% [95% CI: 2.8%, 26.9%], (P =.02) on nicotinamide and 5.2% [−4.2%, 14.6%], (P =.27) on placebo. Vmax ratio improved by 12.6% [5.0%, 20.2%], (P =.002) following nicotinamide, 3.6% [−3.4%, 10.5%], (P =.30) on placebo. A trend for improved visual field mean deviation was observed with 27% improving ≥1 dB on nicotinamide and fewer deteriorating (4%) compared to placebo (P =.02). Conclusions: Nicotinamide supplementation can improve inner retinal function in glaucoma. Further studies underway to elucidate the effects of long‐term nicotinamide supplementation. [ABSTRACT FROM AUTHOR]

Retinal ganglion cells ( RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise-induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain-derived neurotrophic factor ( BDNF) and AMP-activated protein kinase ( AMPK). In middle-aged (12 months old) C57 BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra-ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock-down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement-mediated elimination of synapses. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) must be assembled precisely from 45 protein subunits for it to function correctly. One of its mitochondrial DNA (mtDNA) encoded subunits, ND1, is incorporated during the early stages of complex I assembly. However, little is known about how mutations in ND1 affect this assembly process. We found that in human 143B cybrid cells carrying a homoplasmic MT-ND1 mutation, ND1 protein could not be translated. As a result, the early stages of complex I assembly were disrupted, with mature complex I undetectable and complex I-linked respiration severely reduced to 2.0% of control levels. Interestingly, complex IV (ferrocytochrome c:oxygen oxidoreductase) steady-state levels were also reduced to 40.3%, possibly due to its diminished stability in the absence of respiratory supercomplex formation. This was in comparison with 143B cybrid controls (that contained wild-type mtDNA on the same nuclear background), which exhibited normal complex I, complex IV, and supercomplex assembly. We conclude that the loss of ND1 stalls complex I assembly during the early stages of its biogenesis, which not only results in the loss of mature complex I but also disrupts the stability of complex IV and the respiratory supercomplex to cause mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H2O2) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD. [ABSTRACT FROM AUTHOR]

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

The maternally inherited mitochondrial genome (mtDNA) is present in multimeric form within cells and harbors sequence variants (heteroplasmy). While a single mtDNA variant at high load can cause disease, naturally occurring variants likely persist at low levels across generations of healthy populations. To determine how naturally occurring variants are segregated and transmitted, we generated a mini-pig model, which originates from the same maternal ancestor. Following next-generation sequencing, we identified a series of low-level mtDNA variants in blood samples from the female founder and her daughters. Four variants, ranging from 3% to 20%, were selected for validation by high-resolution melting analysis in 12 tissues from 31 animals across three generations. All four variants were maintained in the offspring, but variant load fluctuated significantly across the generations in several tissues, with sex-specific differences in heart and liver. Moreover, variant load was persistently reduced in high-respiratory organs (heart, brain, diaphragm, and muscle), which correlated significantly with higher mtDNA copy number. However, oocytes showed increased heterogeneity in variant load, which correlated with increased mtDNA copy number during in vitro maturation. Altogether, these outcomes show that naturally occurring mtDNA variants segregate and are maintained in a tissue-specific manner across generations. This segregation likely involves the maintenance of selective mtDNA variants during organogenesis, which can be differentially regulated in oocytes and preimplantation embryos during maturation. [ABSTRACT FROM AUTHOR]

Primary Open Angle Glaucoma (POAG) is a common neurodegenerative disease characterized by the selective and gradual loss of retinal ganglion cells (RGCs). Aging and increased intraocular pressure (IOP) are glaucoma risk factors; nevertheless patients deteriorate at all levels of IOP, implying other causative factors. Recent evidence presents mitochondrial oxidative phosphorylation (OXPHOS) complex-I impairments in POAG. Leber Hereditary Optic Neuropathy (LHON) patients suffer specific and rapid loss of RGCs, predominantly in young adult males, due to complex-I mutations in the mitochondrial genome. This study directly compares the degree of OXPHOS impairment in POAG and LHON patients, testing the hypothesis that the milder clinical disease in POAG is due to a milder complex-I impairment. To assess overall mitochondrial capacity, cells can be forced to produce ATP primarily from mitochondrial OXPHOS by switching the media carbon source to galactose. Under these conditions POAG lymphoblasts grew 1.47 times slower than controls, whilst LHON lymphoblasts demonstrated a greater degree of growth impairment (2.35 times slower). Complex-I enzyme specific activity was reduced by 18% in POAG lymphoblasts and by 29% in LHON lymphoblasts. We also assessed complex-I ATP synthesis, which was 19% decreased in POAG patients and 17% decreased in LHON patients. This study demonstrates both POAG and LHON lymphoblasts have impaired complex-I, and in the majority of aspects the functional defects in POAG were milder than LHON, which could reflect the milder disease development of POAG. This new evidence places POAG in the spectrum of mitochondrial optic neuropathies and raises the possibility for new therapeutic targets aimed at improving mitochondrial function. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is implicated in a growing spectrum of diseases, from neurodegeneration to cancer. Where tissues or transformed cells are available, respirometry and enzymology allow a sophisticated analysis of OXPHOS with modest-cost equipment. The isolation of organelle fractions is also invaluable for determining association of proteins of interest. Here we revisit and consolidate methods to measure whole cell mitochondrial ATP synthesis, respiration, isolation of mitochondria from cultured cells and tissues, and OXPHOS enzymology. We also explain common pitfalls, guide optimisation of the methods for new users, and provide full laboratory protocols in Supplementary materials. [Copyright &y& Elsevier]

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers. [ABSTRACT FROM AUTHOR]

Obesity is associated with chronic low-grade inflammation that contributes to defects in energy metabolism and insulin resistance. Suppressor of cytokine signaling (SOCS)-3 expression is increased in skeletal muscle of obese humans. SOCS3 inhibits leptin signaling in the hypothalamus and insulin signal transduction in adipose tissue and the liver. Skeletal muscle is an important tissue for controlling energy expenditure and whole-body insulin sensitivity; however, the physiological importance of SOCS3 in this tissue has not been examined. Therefore, we generated mice that had SOCS3 specifically deleted in skeletal muscle (SOCS MKO). The SOCS3 MKO mice had normal muscle development, body mass, adiposity, appetite, and energy expenditure compared with wild-type (WT) littermates. Despite similar degrees of obesity when fed a high-fat diet, SOCS3 MKO mice were protected against the development of hyperinsulinemia and insulin resistance because of enhanced skeletal muscle insulin receptor substrate 1 (IRS1) and Akt phosphorylation that resulted in increased skeletal muscle glucose uptake. These data indicate that skeletal muscle SOCS3 does not play a critical role in regulating muscle development or energy expenditure, but it is an important contributing factor for inhibiting insulin sensitivity in obesity. Therapies aimed at inhibiting SOCS3 in skeletal muscle may be effective in reversing obesity-related glucose intolerance and insulin resistance. [ABSTRACT FROM AUTHOR]

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copperII [CuII(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson's disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from CuII(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from CuII(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with CuII(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a CuII(atsm)induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from CuII(atsm). Using these cell culture models our data demon strate that compromised ETC function, due to the absence of O2 as the terminal electron acceptor or dysfunction of individual components of the ETC. is an important determinant in driving the intracellular dissociation of CuII(atsm) that increases cellular retention of the Cu. [ABSTRACT FROM AUTHOR]

Abstract Objective: The high energy demand of the auditory and visual pathways render these sensory systems prone to diseases that impair mitochondrial function. Primary open-angle glaucoma, a neurodegenerative disease of the optic nerve, has recently been associated with a spectrum of mitochondrial abnormalities. This study sought to investigate auditory processing in individuals with open-angle glaucoma. Design/Study sample: Twenty-seven subjects with open-angle glaucoma underwent electrophysiologic (auditory brainstem response), auditory temporal processing (amplitude modulation detection), and speech perception (monosyllabic words in quiet and background noise) assessment in each ear. A cohort of age, gender and hearing level matched control subjects was also tested. Results: While the majority of glaucoma subjects in this study demonstrated normal auditory function, there were a significant number (6/27 subjects, 22%) who showed abnormal auditory brainstem responses and impaired auditory perception in one or both ears. Conclusions: The finding that a significant proportion of subjects with open-angle glaucoma presented with auditory dysfunction provides evidence of systemic neuronal susceptibility. Affected individuals may suffer significant communication difficulties in everyday listening situations. [ABSTRACT FROM AUTHOR]

Abstract: The similarities between glaucoma and mitochondrial optic neuropathies have driven a growing interest in exploring mitochondrial function in glaucoma. The specific loss of retinal ganglion cells is a common feature of mitochondrial diseases – not only the classic mitochondrial optic neuropathies of Leber’s Hereditary Optic Neuropathy and Autosomal Dominant Optic Atrophy – but also occurring together with more severe central nervous system involvement in many other syndromic mitochondrial diseases. The retinal ganglion cell, due to peculiar structural and energetic constraints, appears acutely susceptible to mitochondrial dysfunction. Mitochondrial function is also well known to decline with aging in post-mitotic tissues including neurons. Because age is a risk factor for glaucoma this adds another impetus to investigating mitochondria in this common and heterogeneous neurodegenerative disease. Mitochondrial function may be impaired by either nuclear gene or mitochondrial DNA genetic risk factors, by mechanical stress or chronic hypoperfusion consequent to the commonly raised intraocular pressure in glaucomatous eyes, or by toxic xenobiotic or even light-induced oxidative stress. If primary or secondary mitochondrial dysfunction is further established as contributing to glaucoma pathogenesis, emerging therapies aimed at optimizing mitochondrial function represent potentially exciting new clinical treatments that may slow retinal ganglion cell and vision loss in glaucoma. [Copyright &y& Elsevier]

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies. [ABSTRACT FROM AUTHOR]

Acquired optic neuropathies are a common cause of blindness in adults, and are associated with characteristic morphological changes at the optic nerve head. Accurate and prompt clinical diagnosis, supplemented with imaging where appropriate, is essential to optimize management of the optic neuropathy and to counsel the patient appropriately on its natural history. History taking, optic disc findings, visual field assessment and imaging of the nerve head and surrounding retinal nerve fiber layer are all paramount to achieving the correct diagnosis. This Review highlights the optic nerve head features that are common to the acquired optic neuropathies, and describes the features that can be used to differentiate these various conditions. [ABSTRACT FROM AUTHOR]

Accumulation of neurotoxic amyloid-β (Aβ) is central to the pathology of Alzheimer’s disease (AD). Elucidating the mechanisms of Aβ accumulation will therefore expedite the development of Aβ-targeting AD therapeutics. We examined activity of an Aβ-degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Aβ accumulation by altering Aβ sensitivity to proteolytic degradation. An Aβ amino acid mutation found in familial AD, Aβ interactions with zinc (Zn), and increased Aβ hydrophobicity all strongly prevented Aβ degradation. Consistent to all of these factors is the promotion of specific Aβ aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Aβ accumulation by preventing normal protease activity. Zn also prevented Aβ degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn-induced Aβ amyloid with the metal-protein attenuating compound clioquinol reversed amyloid formation and restored the peptide’s sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Aβ-metal interactions can inhibit Aβ accumulation by restoring the catalytic potential of Aβ-degrading proteases. [ABSTRACT FROM AUTHOR]

Corticosterone induces neuroanatomical and neurochemical changes in the hippocampus that are associated with cognitive impairments. In the present study corticosterone induced cell death in primary hippocampal neurons cultured in Neurobasal + B27 medium. Insulin prevents neuronal cell death induced in a concentration dependent manner. The neuroprotective effect of insulin was reversed by LY294002, a phosphatidylinositol 3′-kinase (PI3 kinase) inhibitor, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059, an upstream blocker of MAPK had no effect. Western blot analyses showed that insulin induced the activation of protein kinase B (Akt). These results suggest that insulin can prevent neuronal cell death induced by corticosterone in hippocampal neurons by modulating the activity of the PI3 kinase/Akt pathway. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Knowledge of the mitochondrial DNA (mtDNA) sequence of divergent murine species is critical from both a phylogenetic perspective and in understanding nuclear-mitochondrial interactions, particularly as the latter influences our xenocybrid models of mitochondrial disease. To this end, the sequence of the mitochondrial genome of the murine species Mus terricolor (formerly Mus dunni) is reported and compared with the published sequence for the common laboratory mouse Mus musculus domesticus strain C57BL/6J. These species are of interest because xenomitochondrial cybrid mice were created that harbor M. terricolor mtDNA in a M. m. domesticus nuclear background. Although the total of 1763 nucleotide substitutions represents striking heterogeneity, the majority of these are silent, leading to highly conserved protein sequences with only 159 amino acid differences. Moreover, 58% of these amino acid differences represented conservative substitutions. All of the tRNA genes and rRNA genes have homology of 91% or greater. The control region shows the greatest heterogeneity, as expected, with 85% homology overall. Regions of 100% homology were found for Conserved Sequence Block I, Conserved Sequence Block III and the L-strand origin of replication. Complex I genes showed the greatest degree of difference among protein-coding genes with amino acid homology of 91–97% among the seven mitochondrial genes. Complexes III and IV genes show high homology ranging from 98–100%. From these data, complex I differences appear most critical for the viability of M. m. domesticus: M. terricolor cybrids. Moreover, the sequence information reported here should be useful in identifying critical regions for mitochondrial transfer between species, for furthering the understanding of mitochondrial dynamics and pathology in transmitochondrial organisms, and for the study of Mus genus origins. [Copyright &y& Elsevier]

By altering key amino acid residues of the Alzheimer's disease- associated amyloid-β peptide, we investigated the mechanism through which amyloid-β inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-β inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-β ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-β oligomeric species. Although the amyloid-βmediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu2+, it was not supported by monovalent Cu+, and experiments with catalase and H2O2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-β-mediated H2O2 production. We propose that amyloid-β-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu2+, and that it may involve the formation of a redox active amyloid-β-methionine radical. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer''s disease (AD) and Parkinson''s disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-β) or protein (α-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-β, α-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils. [Copyright &y& Elsevier]

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-β (Aβ) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Aβ peptide (Aβ1- 42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Aβ1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific "aging" of the Aβ1-42 solution. Maximal COX inhibition occurred when using Aβ1-42 solutions aged for 3-6 h at 30°C. The level of Aβ1-42-mediated COX inhibition increased with aging time up to ∼6 h and then declined progressively with continued aging to 48 h. Photo- induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Aβ as the only Aβ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant Aβ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Aβ is associated with brain mitochondria and that Aβ1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Aβ-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

We have developed a flow cytometric assay to measure the oxidative capacity of cultured lymphoblasts as a possible screening test for patients suspected of having a defect of the mitochondrial respiratory chain. Cells were incubated overnight in serum free media, followed by incubation with dihydroethidium with and without rotenone, and then analysed using flow cytometry to measure fluorescence. Inhibition with rotenone gave an increase in fluorescence compared to uninhibited cells. The change in fluorescence was significantly lower in four of the six patient cell lines, with a correlation between the activity of complex I and change in fluorescence. This method may be applicable to cell lines with defects in other complexes of the respiratory chain. [Copyright &y& Elsevier]

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease. [ABSTRACT FROM AUTHOR]

Abstract: Advancing age is a major risk factor for many neurodegenerative diseases but the underlying pathophysiology is not clear. We hypothesize that aging impairs the ability of neurons in the central nervous system to recover functionally after injury. To test this in retinal ganglion cells in vivo, we developed an optic nerve “stress test” which monitors the functional capacity of the optic nerve and retina, during and after a subischemic injury induced by intraocular pressure elevation. We report that older (18-month) C57BL/6J mice suffered greater loss of inner retinal function compared with younger adult mice following intraocular pressure (IOP) challenge. To investigate whether age-related vulnerability to IOP challenge can be modified, we subjected 12-month-old mice to dietary restriction (DR) (alternate-day fasting) for 6 months. Compared with age-matched ad libitum fed controls, DR mice showed greater recovery in inner retinal function following IOP challenge. DR was associated with reduced oxidative stress level following injury and improved mitochondrial oxidative phosphorylation enzyme activity compared with ad libitum controls. Taken together, this study provides in vivo evidence that DR improves functional recovery of the retina following injury and points to the potential benefits of therapies that target mitochondria for the protection of the aging retina and optic nerve against injury. [Copyright &y& Elsevier]