Mitochondrial absorption of short wavelength light drives primate blue retinal cones into glycolysis which may increase their pace of aging.

Photoreceptors have high energy demands and densely packed mitochondria through which light passes before phototransduction. Old world primates including humans have three cone photoreceptor types mediating color vision with short (S blue), medium (M green), and long (L red) wavelength sensitivities. However, S-cones are enigmatic. They comprise <10% of the total cone population, their responses saturate early, and they are susceptible in aging and disease. Here, we show that primate S-cones actually have few mitochondria and are fueled by glycolysis, not by mitochondrial respiration. Glycolysis has a limited ability to sustain activity, potentially explaining early S-cone saturation. Mitochondria act as optical filters showing reduced light transmission at 400-450 nm where S-cones are most sensitive (420 nm). This absorbance is likely to arise in a mitochondrial porphyrin that absorbs strongly in the Soret band. Hence, reducing mitochondria will improve S-cone sensitivity but result in increased glycolysis as an alternative energy source, potentially increasing diabetic vulnerability due to restricted glucose access. Further, glycolysis carries a price resulting in premature functional decline as seen in aged S-cones. Soret band absorption may also impact on mitochondrial rich M and L cones by reducing sensitivity at the lower end of their spectral sensitivity range resulting in increased differentiation from S-cone responses. These data add to the list of unique characteristic of S-cones and may also explain aspects of their vulnerability.

Amyloid beta deposition and phosphorylated tau accumulation are key features in aged choroidal vessels in the complement factor H knock out model of retinal degeneration.

Extra-cellular deposition including amyloid beta (Aß) is a feature of retinal ageing. It has been documented for Bruch's membrane (BM) where Aß is elevated in complement factor H knockout mice (Cfh(-/-)) proposed as a model for age related macular degeneration. However, arterial deposition in choroidal vessels prior to perfusion across BM has not been examined. Aß is associated with tau phosphorylation and these are linked in blood vessels in Alzheimers Disease where they can drive perivascular pathology. Here we ask if Aß, tau and phosphorylated tau are features of ageing in choroidal vessels in 12 month C57 BL/6 and Cfh(-/-) mice, using immune staining and Western blot analysis. Greater levels of Aß and phosphorylated tau are found in choroidal vessels in Cfh(-/-) mice. Western blot revealed a 40% increase in Aß in Cfh(-/-) over C57 BL/6 mice. Aß deposits coat around 55% of the luminal wall in Cfh(-/-) compared to only about 40% in C57 BL/6. Total tau was similar in both groups, but phosphorylated tau increased by >100% in Cfh(-/-) compared to C57 BL/6 and covered >75% of the luminal wall compared to 50% in C57 BL/6. Hence, phosphorylated tau is a marked choroidal feature in this mouse model. Aß deposition was clumped in Cfh(-/-) mice and likely to influence blood flow dynamics. Disturbed flow is associated with atherogenesis and may be related to the accumulation of membrane attack complex recently identified between choroidal vessels in those at high risk of macular degeneration due to complement factor H polymorphisms.

Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster.

Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflammation with age. Critically, there was a significant increase in average lifespan: 100-175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age.

Biallelic mutations in PLA2G5, encoding group V phospholipase A2, cause benign fleck retina.

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

Mitochondrial decline in the ageing old world primate retina: Little evidence for difference between the centre and periphery.

Mitochondrial decline is a key feature of ageing. The retina has more mitochondria than any other tissue and ages rapidly. To understand human retinal ageing it is critical to examine old world primates that have similar visual systems to humans, and do so across central and peripheral regions, as there is evidence for early central decline. Hence, we examine mitochondrial metrics in young and ageing Macaca fascicularis retinae. In spite of reduced ATP with age, primate mitochondrial complex activity did not decline. But mitochondrial membrane potentials were reduced significantly, and concomitantly, mitochondrial membrane permeability increased. The mitochondrial marker Tom20 declined significantly, consistent with reduced mitochondria number, while VDAC, a voltage dependent anion channel and diffusion pore associated with apoptosis increased significantly. In spite of these clear age-related changes, there was almost no evidence for regional differences between the centre and the periphery in these mitochondrial metrics. Primate cones do not die with age, but many showed marked structural decline with vacuous spaces in proximal inner segments normally occupied by endoplasmic reticulum (ER), that regulate mitochondrial autophagy. In many peripheral cones, ER was displaced by the nucleus that transposed across the outer limiting membrane and could become embedded in mitochondrial populations. These data are consistent with significant changes in retinal mitochondria in old world primate ageing but provide little if any evidence that aged central mitochondria suffer more than those in the periphery.

Do astrocytes respond to light, sound, or electrical stimulation?

Astrocytes are not only the most populous cell type in the human brain, but they also have the most extensive and diverse sets of connections, across synapses, axons, blood vessels, as well as having their own internal network. Unsurprisingly, they are associated with many brain functions; from the synaptic transmission to energy metabolism and fluid homeostasis, and from cerebral blood flow and blood-brain barrier maintenance to neuroprotection, memory, immune defenses and detoxification, sleep, and early development. And yet, notwithstanding these key roles, so many current therapeutic approaches to a range of brain disorders have largely neglected their potential involvement. In this review, we consider the role of astrocytes in three brain therapies; two are emerging treatments (photobiomodulation and ultrasound), while the other is well-established (deep brain stimulation). In essence, we explore the issue of whether external sources, such as light, sound, or electricity, can influence the function of astrocytes, as they do neurons. We find that, when taken all together, each of these external sources can influence many, if not, all of the functions associated with astrocytes. These include influencing neuronal activity, prompting neuroprotection, reducing inflammation (astrogliosis) and potentially increasing cerebral blood flow and stimulating the glymphatic system. We suggest that astrocytes, just like neurons, can respond positively to each of these external applications and that their activation could each impart many beneficial outcomes on brain function; they are likely to be key players underpinning the mechanisms behind many therapeutic strategies.

From the Lab to the Field: Translating Applications of Near-Infrared Light from Laboratory to the Field to Improve Honeybee Mitochondrial Function and Hive Health.

Objective: Bee populations are under threat from diverse sources from climate change to insecticide use. These culminate in physiological stress undermining mitochondrial function. In laboratory environments, mitochondrial stress can be ameliorated by long wavelength light that protects insects individually against stress. In this study, we ask if these results can be translated to large insect communities and complex environments in the form of field honeybee hives. Materials and methods: We embed 670 nm light devices into honeybee hives in the field, and in sampled populations measure mitochondrial function, resistance to insecticide exposure, and the maintenance of hive temperatures in challenging summer conditions. Results: We show that 670 nm light increases the mitochondrial function and protects bees when they are exposed to imidacloprid in the winter supplementary feed. Hives with 670 nm lights maintained stable temperatures compared with controls in adverse weather conditions. Conclusions: This proof-of-principal study opens the door to widespread use of long wavelength light to protect honeybee hives from the increasing threats undermining their physiology that can cause colony collapse.

Mitochondria are specifically vulnerable to 420nm light in drosophila which undermines their function and is associated with reduced fly mobility.

Increased blue light exposure has become a matter of concern as it has a range of detrimental effects, but the mechanisms remain unclear. Mitochondria absorb short wavelength light but have a specific absorbance at 420nm at the lower end of the human visual range. This 420nm absorption is probably due to the presence of porphyrin. We examine the impact of 420nm exposure on drosophila melanogaster mitochondria and its impact on fly mobility. Daily 15 mins exposures for a week significantly reduced mitochondrial complex activities and increased mitochondrial inner membrane permeability, which is a key metric of mitochondrial health. Adenosine triphosphate (ATP) levels were not significantly reduced and mobility was unchanged. There are multiple options for energy/time exposure combinations, but we then applied single 420nm exposure of 3h to increase the probability of an effect on ATP and mobility, and both were significantly reduced. ATP and mitochondrial membrane permeability recovered and over corrected at 72h post exposure. However, despite this, normal mobility did not return. Hence, the effect of short wavelengths on mitochondrial function is to reduce complex activity and increasing membrane permeability, but light exposure to reduce ATP and to translate into reduced mobility needs to be sustained.

The 3D organisation of mitochondria in primate photoreceptors.

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.

Complement factor h is critical in the maintenance of retinal perfusion.

Vascular pathologies are known to be associated with age-related macular degeneration. Recently, age-related macular degeneration was associated with a single-nucleotide substitution of the complement factor H (CFH) gene, part of the alternative pathway of the complement system, a critical element in the innate immune response. Such polymorphisms are found in more than 50% of cases of age-related macular degeneration. Here we show that the absence of CFH causes an autoimmune response that targets the vascular endothelium of both the inner and outer retinal vascular networks. In CFH-knockout (cfh(-/-)) mice, C3 and C3b, key components of the complement system, are progressively deposited on retinal vessels, which subsequently become restricted and wither, resulting in a reduction of retinal blood supply. This result leads to increased oxygen stress. While such effects are not systemic, these structural changes are mirrored in functional changes with a substantial decline in retinal blood flow dynamics. When the system is challenged functionally by laser-induced choroidal neovascularization, fluorescein leakage was significantly smaller in cfh(-/-) mice compared with controls, likely due to reduced retinal perfusion. These data reveal that in both the presence and absence of exogenous challenge to the innate immune system, CFH is required to maintain normal levels of retinal perfusion. It is likely that C3 and C3b accumulation in the aged CFH-deficient retina is associated with complement-mediated retinal endothelium destruction.

Complement factor H regulates retinal development and its absence may establish a footprint for age related macular degeneration.

Age related macular degeneration (AMD) is the most common blinding disease in those over 60 years. In 50% of cases it is associated with polymorphisms of complement factor H (FH), implicating immune vulnerability. But such individuals may exhibit abnormal outer retinal blood flow decades before disease initiation, suggesting an early disease footprint. FH is expressed in the retinal pigmented epithelium (RPE). During development the RPE is adjacent to the site of retinal mitosis and complex regulatory interactions occur between the relatively mature RPE and retinal neuronal precursors that control the cell cycle. Here we ask if the absence of FH from the RPE influences retinal development using a mouse CFH knockout (Cfh-/-) with an aged retinal degenerative phenotype. We reveal that from birth, these mice have significantly disrupted and delayed retinal development. However, once development is complete, their retinae appear relatively normal, although many photoreceptor and RPE mitochondria are abnormally large, suggesting dysfunction consistent with premature ATP decline in Cfh-/-. Total retinal mtDNA is also reduced and these deficits are associated shortly after with reduced retinal function. Cfh-/+ mice also show significant abnormal patterns of cell production but not as great as in Cfh-/-. These results reveal that not only is FH an important player in sculpting retinal development but also that the developmental abnormality in Cfh-/- likely establishes critical vulnerability for later aged retinal degeneration.

Fundamental differences in patterns of retinal ageing between primates and mice.

Photoreceptors have high metabolic demands and age rapidly, undermining visual function. We base our understanding mainly on ageing mice where elevated inflammation, extracellular deposition, including that of amyloid beta, and rod and cone photoreceptor loss occur, but cones are not lost in ageing primate although their function declines, revealing that primate and mouse age differently. We examine ageing primate retinae and show elevated stress but low inflammation. However, aged primates have a >70% reduction in adenosine triphosphate (ATP) and a decrease in cytochrome c oxidase. There is a shift in cone mitochondrial positioning and glycolytic activity increases. Bruch's membrane thickens but unlike in mice, amyloid beta is absent. Hence, reduced ATP may explain cone functional decline in ageing but their retained presence offers the possibility of functional restoration if they can be fuelled appropriately to restore cellular function. This is important because as humans we largely depend on cone function to see and are rarely fully dark adapted. Presence of limited aged inflammation and amyloid beta deposition question some of the therapeutic approaches taken to resolve problems of retinal ageing in humans and the possible lack of success in clinical trials in macular degeneration that have targeted inflammatory agents.

A day in the life of mitochondria reveals shifting workloads.

Mitochondria provide energy for cellular function. We examine daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity. These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health.

Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.

PURPOSE: To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats. METHODS: Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67. RESULTS: In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells). CONCLUSIONS: Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

Photobiomodulation reduces gliosis in the basal ganglia of aged mice.

This study explored the effects of long-term photobiomodulation (PBM) on the glial and neuronal organization in the striatum of aged mice. Mice aged 12 months were pretreated with PBM (670 nm) for 20 minutes per day, commencing at 5 months old and continued for 8 months. We had 2 control groups, young at 3 months and aged at 12 months old; these mice received no treatment. Brains were aldehyde-fixed and processed for immunohistochemistry with various glial and neuronal markers. We found a clear reduction in glial cell number, both astrocytes and microglia, in the striatum after PBM in aged mice. By contrast, the number of 2 types of striatal interneurons (parvalbumin+ and encephalopsin+), together with the density of striatal dopaminergic terminals (and their midbrain cell bodies), remained unchanged after such treatment. In summary, our results indicated that long-term PBM had beneficial effects on the aging striatum by reducing glial cell number; and furthermore, that this treatment did not have any deleterious effects on the neurons and terminations in this nucleus.

To unite or divide: mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss.

Mitochondrial function declines with age and is associated with age-related disorders and cell death. In the retina this is critical as photoreceptor energy demands are the greatest in the body and aged cell loss large (~30%). But mitochondria can fuse or divide to accommodate changing demands. We explore ageing mitochondrial dynamics in young (1 month) and old (12 months) mouse retina, investigating changes in mitochondrial fission (Fis1) and fusion (Opa1) proteins, cytochrome C oxidase (COX III), which reflects mitochondrial metabolic status, and heat shock protein 60 (Hsp60) that is a mitochondrial chaperon for protein folding.Western blots showed each protein declined with age. However, within this, immunostaining revealed increases of around 50% in Fis1 and Opa1 in photoreceptor inner segments (IS). Electron microscope analysis revealed mitochondrial fragmentation with age and marked changes in morphology in IS, consistent with elevated dynamics. COX III declined by approximately 30% in IS, but Hsp60 reductions were around 80% in the outer plexiform layer.Our results are consistent with declining mitochondrial metabolism. But also with increased photoreceptor mitochondrial dynamics that differ from other retinal regions, perhaps reflecting attempts to maintain function. These changes are the platform for age related photoreceptor loss initiated after 12 months.

Mitochondrial decline precedes phenotype development in the complement factor H mouse model of retinal degeneration but can be corrected by near infrared light.

Mitochondria produce adenosine triphosphate (ATP), critical for cellular metabolism. ATP declines with age, which is associated with inflammation. Here, we measure retinal and brain ATP in normal C57BL/6 and complement factor H knockout mice (Cfh(-/-)), which are proposed as a model of age-related macular degeneration. We show a significant premature 30% decline in retinal ATP in Cfh(-/-) mice and a subsequent shift in expression of a heat shock protein that is predominantly mitochondrial (Hsp60). Changes in Hsp60 are associated with stress and neuroprotection. We find no differences in brain ATP between C57BL/6 and Cfh(-/-) mice. Near infrared (NIR) increases ATP and reduces inflammation. ATP decline in Cfh(-/-) mice was corrected with NIR which also shifted Hsp60 labeling patterns. ATP decline in Cfh(-/-) mice occurs before inflammation becomes established and photoreceptor loss occurs and may relate to disease etiology. However, ATP levels were corrected with NIR. In summary, we provide evidence for a mitochondrial basis for this disease in mice and correct this with simple light exposure known to improve mitochondrial function.