Distinctive clinical and histological characteristics of atrophic and hypertrophic facial photoageing.

BACKGROUND: Photoageing describes complex cutaneous changes which occur following chronic exposure to solar ultraviolet radiation (UVR). Amongst White Northern Europeans, facial photoageing appears as distinct clinical phenotypes: 'hypertrophic' photoageing (HP) and 'atrophic' photoageing (AP). Deep, coarse wrinkles predominate in individuals with HP, whereas those with AP have relatively smooth, unwrinkled skin with pronounced telangiectasia. AP individuals have an increased propensity for developing keratinocyte cancers. OBJECTIVES: To investigate whether histological differences underlie these distinct phenotypes of facial photoageing. METHODS: Facial skin biopsies were obtained from participants with AP (10 M, 10 F; mean age: 78.7 years) or HP (10 M, 10 F; mean age: 74.5 years) and were assessed histologically and by immunohistochemistry. RESULTS: Demographic characterization revealed 95% of AP subjects, as compared to 35% with HP, were Fitzpatrick skin type I/II; of these, 50% had a history of one or more keratinocyte cancers. There was no history of keratinocyte cancers in the HP cohort. Analysis of UVR-induced mitochondrial DNA damage confirmed that all volunteers had received similar lifetime cumulative doses of sun exposure. Histologically, male AP had a significantly thicker epidermis than did AP females or those of either sex with HP. HP facial skin exhibited severe solar elastosis, whereas in AP facial skin, solar elastosis was apparent only in females. Loss of papillary dermal fibrillin-rich microfibrils occurred in all HP and AP female subjects, but not in AP males. Furthermore, male AP had a significant reduction in collagen VII at the dermal-epidermal junction than did AP females or those of either sex with HP. CONCLUSIONS: This study provides further evidence that AP and HP represent distinct clinical and histological entities. Knowledge of these two phenotypes is clinically relevant due to the increased prevalence of keratinocyte cancers in those - particularly males - with the AP phenotype.

In vitro protective effects of an aqueous extract of Clitoria ternatea L. flower against hydrogen peroxide-induced cytotoxicity and UV-induced mtDNA damage in human keratinocytes.

The traditional practice of eating the flowers of Clitoria ternatea L. or drinking their infusion as herbal tea in some of the Asian countries is believed to promote a younger skin complexion and defend against skin aging. This study was conducted to investigate the protective effect of C. ternatea flower water extract (CTW) against hydrogen peroxide-induced cytotoxicity and ultraviolet (UV)-induced mitochondrial DNA (mtDNA) damage in human keratinocytes. The protective effect against hydrogen peroxide-induced cytotoxicity was determined by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and mtDNA damage induced by UV was determined by polymerase chain reaction. Preincubation of HaCaT with 100, 250, and 500 µg/ml CTW reduced cytotoxicity effects of H2 O2 compared with control (H2 O2 alone). CTW also significantly reduced mtDNA damage in UV-exposed HaCaT (p < .05). CTW was chemically-characterized using high resolution liquid chromatography-mass spectrometry. The main compounds detected were assigned as anthocyanins derived from delphinidin, including polyacylated ternatins, and flavonol glycosides derived from quercetin and kaempferol. These results demonstrated the protective effects of C. ternatea flower extracts that contain polyacylated anthocyanins and flavonol glycosides as major constituents, against H2 O2 and UV-induced oxidative stress on skin cells, and may provide some explanation for the putative traditional and cosmetic uses of C. ternatea flower against skin aging.

Oxidative stress and ageing.

Oxidative stress is the resultant damage due to redox imbalances (increase in destructive free radicals [reactive oxygen species (ROS)] and reduction in antioxidant protection/pathways) and is linked to ageing in many tissues including skin. In ageing skin there are bioenergetic differences between keratinocytes and fibroblasts which provide a potential ageing biomarker. The differences in skin bioenergy are part of the mitochondrial theory of ageing which remains one of the most widely accepted ageing theories describing subsequent increasing free radical generation. Mitochondria are the major source of cellular oxidative stress and form part of the vicious cycle theory of ageing. External and internal sources of oxidative stress include UVR/IR, pollution (environment), lifestyle (exercise and diet), alcohol and smoking all of which may potentially impact on skin although many exogenous actives and endogenous antioxidant defence systems have been described to help abrogate the increased stress. This also links to differences in skin cell types in terms of the UVR action spectrum for nuclear and mitochondrial DNA damage (the latter a previously described UVR biomarker in skin). Recent work associates bioenergy production and oxidative stress with pigment production thereby providing another additional potential avenue for targeted anti-ageing intervention in skin. This new data supporting the detrimental effects of the numerous wavelengths of UVR may aid in the development of cosmetic/sunscreen design to reduce the effects of photoageing. Recently, complex II of the mitochondrial electron transport chain appears to be more important than previously thought in the generation of free radicals (suggested predominantly by non-human studies). We investigated the relationship between complex II and ageing using human skin as a model tissue. The rate of complex II activity per unit of mitochondria was determined in fibroblasts and keratinocytes cultured from skin covering a wide age range. Complex II activity significantly decreased with age in fibroblasts (P = 0·015), but not in keratinocytes. This was associated with a significant decline in transcript expression (P = 0·008 and P = 0·001) and protein levels (P = 0·0006 and P = 0·005) of the SDHA and SDHB catalytic subunits of complex II respectively. In addition there was a significant decrease in complex II activity with age (P = 0·029) that was specific to senescent skin cells, our study being the first to investigate these differences with senescence and skin age. There was no decrease in complex IV activity with increasing age, suggesting possible locality to complex II. Our study provides a future potential biomarker for monitoring the progression of skin ageing.

Oxidative stress--a key emerging impact factor in health, ageing, lifestyle and aesthetics.

Oxidative stress is the resultant damage that arises due to redox imbalances, more specifically an increase in destructive free radicals and reduction in protection from antioxidants and the antioxidant defence pathways. Oxidation of lipids by reactive oxygen species (ROS) can damage cellular structures and result in premature cell death. At low levels, ROS-induced oxidative stress can be prevented through the action of antioxidants, however, when ROS are present in excess, inflammation and cytotoxicity eventually results leading to cellular oxidative stress damage. Increasing evidence for the role of oxidative stress in various diseases including neurological, dermatological, and cardiovascular diseases is now emerging. Mitochondria are the principal source (90%) of ROS in the cell, with superoxide radicals being generated when molecular oxygen is combined with free electrons. Given the key role of mitochondria in the generation of cellular oxidative stress it is worth considering this organelle and the process in more detail and to provide methods of intervention.

Mitochondrial DNA damage as a biomarker for ultraviolet radiation exposure and oxidative stress.

The skin is regularly exposed to the harmful effects of sunlight, such as ultraviolet radiation (UVR), which leads to ageing effects as well as clinical precancerous lesions and skin cancer. The accumulation of mitochondrial DNA (mtDNA) damage has been strongly associated as an underlying cause of the general ageing process in tissues and mtDNA damage has been associated with cancer development in many tissues including human skin. This scenario is linked to the key roles of mitochondrial function and mtDNA both in terms of energy production and also oxidative stress production as well as a mediator of apoptosis. We and others have pioneered the use of mtDNA damage as a highly sensitive biomarker of UVR exposure and oxidative stress in human skin; furthermore, ageing-dependent mtDNA mutations can be accelerated by exposure to sunlight. In addition, this review will also highlight useful applications of mtDNA as a biomarker of UVR-induced oxidative stress including effects of antioxidants.

Increasing the therapeutic efficacy of docetaxel for cutaneous squamous cell carcinoma through the combined inhibition of phosphatidylinositol 3-kinase/AKT signalling and autophagy.

Early-stage cutaneous squamous cell carcinoma (cSCC) has a favourable prognosis. Metastatic disease is probably associated with chemoresistance mediated through the activation of pro-survival phosphatidylinositol 3-kinase/AKT signalling. Inhibition of activated AKT partially increases chemosensitivity but induces autophagy, the principal lysosomal mechanism for the bulk degradation and recycling of proteins and damaged organelles. The aim of the current study was to test the hypothesis that combined inhibition of AKT signalling and autophagy by the lysosomal inhibitor chloroquine increases the susceptibility to docetaxel-induced apoptosis of cSCC cells isolated from a lymph-node metastasis. Combined AKT inhibition and chloroquine treatment of MET 4 cSCC cells resulted in significantly enhanced inhibition of cell viability and apoptosis induced by clinically achievable concentrations of docetaxel (P < 0.001). Inhibition of both autophagy and AKT thus represents an effective and viable therapeutic strategy to increase the cytotoxicity of docetaxel for the treatment of advanced cSCC.

Tomato paste rich in lycopene protects against cutaneous photodamage in humans in vivo: a randomized controlled trial.

BACKGROUND: Previous epidemiological, animal and human data report that lycopene has a protective effect against ultraviolet radiation (UVR)-induced erythema. OBJECTIVES: We examined whether tomato paste--rich in lycopene, a powerful antioxidant--can protect human skin against UVR-induced effects partially mediated by oxidative stress, i.e. erythema, matrix changes and mitochondrial DNA (mtDNA) damage. METHODS: In a randomized controlled study, 20 healthy women (median age 33 years, range 21-47; phototype I/II) ingested 55 g tomato paste (16 mg lycopene) in olive oil, or olive oil alone, daily for 12 weeks. Pre- and postsupplementation, UVR erythemal sensitivity was assessed visually as the minimal erythema dose (MED) and quantified with a reflectance instrument. Biopsies were taken from unexposed and UVR-exposed (3 × MED 24 h earlier) buttock skin pre- and postsupplementation, and analysed immunohistochemically for procollagen (pC) I, fibrillin-1 and matrix metalloproteinase (MMP)-1, and by quantitative polymerase chain reaction for mtDNA 3895-bp deletion. RESULTS: Mean ± SD erythemal D(30) was significantly higher following tomato paste vs. control (baseline, 26·5 ± 7·5 mJ cm(-2); control, 23 ± 6·6 mJ cm(-2); tomato paste, 36·6 ± 14·7 mJ cm(-2); P = 0·03), while the MED was not significantly different between groups (baseline, 35·1 ± 9·9 mJ cm(-2); control, 32·6 ± 9·6 mJ cm(-2); tomato paste, 42·2 ± 11·3 mJ cm(-2)). Presupplementation, UVR induced an increase in MMP-1 (P = 0·01) and a reduction in fibrillin-1 (P = 0·03). Postsupplementation, UVR-induced MMP-1 was reduced in the tomato paste vs. control group (P = 0·04), while the UVR-induced reduction in fibrillin-1 was similarly abrogated in both groups, and an increase in pCI deposition was seen following tomato paste (P = 0·05). mtDNA 3895-bp deletion following 3 × MED UVR was significantly reduced postsupplementation with tomato paste (P = 0·01). CONCLUSIONS: Tomato paste containing lycopene provides protection against acute and potentially longer-term aspects of photodamage.

Real-time polymerase chain reaction analysis of a 3895-bp mitochondrial DNA deletion in epithelial swabs and its use as a quantitative marker for sunlight exposure in human skin.

BACKGROUND: The use of mitochondrial DNA (mtDNA) damage as a reliable and highly sensitive biomarker of ultraviolet (UV) radiation exposure in both the dermis and epidermis has now been well developed by our group and others. We have previously identified a 3895-bp mtDNA deletion which occurred more frequently and to a higher level in usually sun-exposed skin as opposed to occasionally sun-exposed skin. This work focused on older-aged individuals and, in particular, perilesional, histologically normal skin biopsies taken from patients with skin cancer. OBJECTIVES: To develop novel, less-invasive methods of obtaining skin samples (i.e. epidermis) from volunteers covering a much wider age range and larger number of individuals (n = 239). METHODS: The 3895-bp deletion was quantified by a specific real-time polymerase chain reaction assay in normal human epidermis samples taken from three body sites with differing sun exposure. RESULTS: The results show a statistical increase of the level of the 3895-bp deletion with increasing sun exposure in the epidermal swabs of human skin (P < 0·001) and with increasing age of the donor in the needle biopsy samples. CONCLUSIONS: These data suggest that the upper layers of the epidermis are an accessible and reliable site for assessing mtDNA damage caused by UV exposure.

Management of field change in actinic keratosis.

Field cancerization was first described in 1953 as histologically altered mucosa surrounding tumours removed from the upper gastro-intestinal tract. Over the years the definition has changed to include an area which is clinically occult but has multifocal preneoplastic changes, showing genetic mutations and which precedes the development of second primary tumours and local recurrences. Field cancerization has been described in the oropharynx, oesophagus, stomach, lung, colon, anus, cervix, bladder and skin. Various molecular techniques have been developed to look for genetic mutations and clonality in areas of field change. These studies have highlighted the need for early detection and treatment in order to prevent the development of tumours and local recurrences. In this article we examine the concept of field cancerization and treatments available to manage field change.

Direct monitoring of UV-induced free radical generation in HaCaT keratinocytes.

BACKGROUND: Ultraviolet radiation (UVR) is one of the most important aetiological factors in the development of skin cancer, with an estimated 100,000 new cases of nonmelanoma skin cancer (NMSC) diagnosed each year in the UK. To date, little work has been carried out to investigate the role of UVR in the increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) following exposure of skin cells to simulated solar UVR. AIM: To monitor directly the effects of simulated solar UVR on ROS and RNS generation in HaCaT keratinocytes. METHODS: This study reports the use of electrochemical monitoring techniques for the direct, real-time detection of two highly reactive free radical species, superoxide (O2-) and nitric oxide (NO), from HaCaT keratinocyte cells that had been exposed to a source of UVR designed to simulate the doses of UVA and UVB found in solar light. RESULTS: An increase in both O2- and NO generation was observed in HaCaT cells that had been exposed to UVR. No detectable increase in either species was observed in cells that had not been exposed to UVR. The specificity of the electrochemical methods for O2- or NO was confirmed through the scavenging or inhibition of these species. CONCLUSION: The findings of this study demonstrated that exposure of HaCaT cells to relatively low doses of UVR resulted in the immediate generation of both O2- and NO, therefore potentially leading to the downstream generation of highly damaging metabolites and the development of a number of pathologies, including cancer.

The role of mitochondria in ageing and carcinogenesis.

Mitochondria can perform multiple cellular functions including energy production, cell proliferation and apoptosis. These organelles contain their own genetic material, mitochondrial DNA (mtDNA), which is maternally inherited. Although much smaller than the nuclear genome, mtDNA is equally important, as it has been hypothesized to play a crucial role in ageing and carcinogenesis. This is partly due to the fact that mitochondria represent the major site for the generation of cellular oxidative stress and play a key role in mediating programmed cell death (apoptosis). Damage to mtDNA is therefore an important contributor to human ageing, cancer and neurodegenerative diseases. The most relevant footprints of mtDNA damage are point mutations of single bases, or deletions of the 16.5-kb mitochondrial genome. This review will focus on the key roles of mitochondrial function and mtDNA in oxidative stress production and as a mediator of apoptosis, and on the use of mtDNA as a biomarker of sun exposure. This will be related to the contribution of mitochondria and mtDNA in the ageing process and cancer, with a specific focus on human skin. In conclusion, it is likely that the interplay between nuclear and mitochondrial genes may hold the final understanding of the mitochondrial role in these disease processes.

Real-time PCR analysis of a 3895 bp mitochondrial DNA deletion in nonmelanoma skin cancer and its use as a quantitative marker for sunlight exposure in human skin.

Previous findings from our own laboratory have shown that the frequency of occurrence (i.e. the simple presence or absence) of the 3895 bp mitochondrial DNA deletion is increased with increasing sun exposure. The present study has significantly extended this work by developing, validating and then using a quantitative real-time PCR assay to investigate for the first time the actual level (as opposed to the frequency of occurrence) of the 3895 bp deletion in human skin from different sun-exposed body sites and tumours from nonmelanoma skin cancer patients. We investigated the 3895 bp deletion in 104 age-matched split human skin samples taken from various sun-exposed sites defined as usually exposed (n = 60) and occasionally exposed (n = 44) when outdoors. The results clearly show an increased level of the 3895 bp deletion with increasing sun exposure. Specifically, there was a significantly higher level of the deletion in the usually sun-exposed compared to the occasionally sun-exposed skin (P = 0.0009 for dermis, P = 0.008 for epidermis; two-tailed t-test). Our study has also extended previous findings by showing that the level of the 3895 bp deletion is significantly higher in the dermis compared with the epidermis both in the occasionally sun-exposed samples (P = 0.0143) and in the usually sun-exposed skin. (P = 0.0007).

Mitochondrial DNA damage in non-melanoma skin cancer.

Mitochondrial DNA (mtDNA) damage, predominantly encompassing point mutations, has been reported in a variety of cancers. Here we present in human skin, the first detailed study of the distribution of multiple forms of mtDNA damage in nonmelanoma skin cancer (NMSC) compared to histologically normal perilesional dermis and epidermis. We present the first entire spectrum of deletions found between different types of skin tumours and perilesional skin. In addition, we provide the first quantitative data for the incidence of the common deletion as well as the first report of specific tandem duplications in tumours from any tissue. Importantly, this work shows that there are clear differences in the distribution of deletions between the tumour and the histologically normal perilesional skin. Furthermore, DNA sequencing of four mutation 'hotspot' regions of the mitochondrial genome identified a previously unreported somatic heteroplasmic mutation in an SCC patient. In addition, 81 unreported and reported homoplasmic single base changes were identified in the other NMSC patients. Unlike the distribution of deletions and the heteroplasmic mutation, these homoplasmic mutations were present in both tumour and perilesional skin, which suggests that for some genetic studies the traditional use of histologically normal perilesional skin from NMSC patients may be limited. Currently, it is unclear whether mtDNA damage has a direct link to skin cancer or it may simply reflect an underlying nuclear DNA instability.

The relation between melanocortin 1 receptor genotype and experimentally assessed ultraviolet radiation sensitivity.

Pigmentary phenotype is a key determinant of an individual's response to ultraviolet radiation with the presence of phaeomelanin thought to be of particular importance. Reports of minimal erythema testing, however, have failed to show a consistent difference between skin type I and other skin types. The melanocortin 1 receptor is a key genetic determinant of the cutaneous response to ultraviolet radiation. In this study we investigate the relation between experimentally induced erythemal response to ultraviolet radiation and the melanocortin 1 receptor genotype. Phototesting was performed in 20 redheads and 20 nonredheaded subjects, the majority of whom were also screened for the presence of melanocortin 1 receptor variants. The majority of redheads sequenced (89%) had two melanocortin 1 receptor variants previously found to be associated with red hair compared to none of the controls. There was no significant difference between the groups in minimal erythema dose: the median minimal erythema dose in redheads was 44 mJ per cm2 (interquartile range 34-56) and in the nonredheaded group was 40 mJ per cm2 (interquartile range 40-56). Objective measurements of ultraviolet-B-induced erythema were performed using reflectance instrument measurements of erythema intensity and dose-response curves constructed for each subject. The slope of the dose-response curve in the redheaded group was statistically greater than in the nonredheaded group (median in redheads 4.08 vs 3.56 for controls, 95% confidence interval for the difference between the medians being 0.01-1.23, p = 0.043). In addition the ratio D0.05:D0.025 was significantly lower for the redheaded group (median in redheads 1.22, interquartile range 1.18-1.26; median in nonreds 1.28, interquartile range 1.23-1.32; p < 0.05). Thus, although the minimal erythema dose values were not different, subjects with red hair develop greater intensity of erythema than nonredheaded individuals when doses greater than the minimal erythema dose are given. Importantly, when analyzed by genotype alone rather than phenotype, the slope of the erythema dose-response differed between those persons who were homozygous or heterozygous mutants and wildtype/pseudo-wildtype (p = 0.026).

Expression, candidate gene, and population studies of the melanocortin 5 receptor.

In mouse the melanocortin 5 receptor is known to regulate sebaceous gland function. To clarify its role in man, we have studied melanocortin 5 receptor expression in skin, and allelic variation at the melanocortin 5 receptor locus in diverse human populations and candidate disease groups. Melanocortin 5 receptor protein and mRNA expression were studied by immunohistochemistry and reverse transcriptase polymerase chain reaction. Melanocortin 5 receptor mRNA was detected in normal skin and cultured keratinocytes but not in cultured fibroblasts or melanocytes. Immunohistochemistry revealed melanocortin 5 receptor immunoreactivity in the epithelium and appendages, including the sebaceous gland, eccrine glands, and apocrine glands, as well as low level expression in the interfollciular epidermis. In order to screen for genetic diversity in the melanocortin 5 receptor that might be useful for allelic association studies we sequenced the entire melanocortin 5 receptor coding region in a range of human populations. One nonsynonymous change (Phe209Leu) and four synonymous changes (Ala81Ala, Asp108Asp, Ser125Ser, and Thr248Thr) were identified. Similar results were found in each of the populations except for the Inuit in which only the Asp108Asp variant was seen. The apparent "global distribution" of melanocortin 5 receptor variants may indicate that they are old in evolutionary terms. Variation of melanocortin 5 receptor was examined in patients with acne (n = 21), hidradenitis supprativa (n = 4), and sebaceous gland lesions comprising sebaceous nevi, adenomas, and hyperplasia (n = 13). No additional mutations were found. In order to determine the functional status of the Phe209Leu change, increase in cAMP in response to stimulation with alpha-melanocyte-stimulating hormone was measured in HEK-293 cells transfected with either wild-type or the Phe209Leu variant. The variant melanocortin 5 receptor was shown to act in a concentration-dependent manner, which did not differ from that of wild type. We have therefore found no evidence of a causative role for melanocortin 5 receptor in sebaceous gland dysfunction, and in the absence of any association between variation at the locus and disease group, the pathophysiologic role of the melanocortin 5 receptor in man requires further study.

Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation.

Variants of the melanocortin 1 receptor (MC1R) gene are common in individuals with red hair and fair skin, but the relative contribution to these pigmentary traits in heterozygotes, homozygotes and compound heterozygotes for variants at this locus from the multiple alleles present in Caucasian populations is unclear. We have investigated 174 individuals from 11 large kindreds with a preponderance of red hair and an additional 99 unrelated redheads, for MC1R variants and have confirmed that red hair is usually inherited as a recessive characteristic with the R151C, R160W, D294H, R142H, 86insA and 537insC alleles at this locus. The V60L variant, which is common in the population may act as a partially penetrant recessive allele. These individuals plus 167 randomly ascertained Caucasians demonstrate that heterozygotes for two alleles, R151C and 537insC, have a significantly elevated risk of red hair. The shade of red hair frequently differs in heterozygotes from that in homozygotes/compound heterozygotes and there is also evidence for a heterozygote effect on beard hair colour, skin type and freckling. The data provide evidence for a dosage effect of MC1R variants on hair as well as skin colour.

The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin.

We and colleagues have suggested that deletions of mitochondrial DNA may be useful as a biomarker of ultraviolet radiation exposure in skin. In this study using a southwestern approach involving monoclonal antibodies against thymine dimers we provide direct evidence for the presence of ultraviolet-induced damage in mitochondrial DNA purified from any nuclear DNA contamination. Previous studies have been limited, as they have focused on the frequency of a single mitochondrial DNA deletion. Therefore we have addressed the question of the spectrum of mitochondrial DNA deletions in skin and whether this can be used as an index of overall DNA damage. We have used a long polymerase chain reaction technique to determine the mitochondrial DNA deletion spectrum of almost the entire mitochondrial genome in 71 split skin samples in relation to sun exposure. There was a significant increase in the number of deletions with increasing ultraviolet exposure in the epidermis (Kruskal-Wallis test, p = 0.0015) but not the dermis (p = 0.6376). The findings in the epidermis are not confounded by any age-dependent increases in mitochondrial DNA deletions also detected by the long polymerase chain reaction technique. The large spectrum of deletions identified in our study highlights the ubiquitous nature and the high mutational load of mitochondrial DNA associated with ultraviolet exposure and chronologic aging. Compared with the detection of single deletions using competitive polymerase chain reaction, we show that long polymerase chain reaction is a sensitive technique and may therefore provide a more comprehensive, although not quantitative, index of overall mitochondrial DNA damage in skin.

Late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene.

Genetic defects affecting the mitochondrial respiratory chain are an important cause of neurological disease. Previously, we identified a family with complex II deficiency and late-onset neurodegenerative disease with progressive optic atrophy, ataxia, and myopathy. The affected family members are now shown to carry a C-to-T transition in one allele of the nuclear gene encoding the flavoprotein subunit of complex II. Mutation of the equivalent base in Escherichia coli generates an inactive enzyme unable to bind flavin adenine dinucleotide covalently. Compatible with these findings, our patients have an approximate 50% decrease in complex II and succinate dehydrogenase activity. These results suggest that genetic defects of nuclear-encoded subunits of the mitochondrial respiratory chain can result in late-onset neurodegenerative disease.