Influence of motivation and academic performance in the use of Augmented Reality in education. A systematic review.

The recent technologies rise today as a tool of significant importance today, especially in the educational context. In this sense, Augmented Reality (AR) is a technology that is achieving a greater presence in educational centers in the last decade. However, Augmented Reality has not been explored in depth at the Secondary Education stage. Due to this, it is essential to analyze and concentrate the scientific research developed around this educational technology at that stage. Therefore, the aim of this research is to describe the influence that Augmented Reality shows on the motivation and academic performance of students in the Secondary Education stage. In relation to the methodology, a systematic review of the literature has been conducted using the Kitchenham protocol, where several factors have been analyzed, such as subjects, activities, and electronic implementation devices, together with the effects on motivation and student's academic performance. The Scopus and Web of Science (WoS) databases have been used to search for scientific papers, with a total of 344 investigations being analyzed between 2012 and 2022. The methodological stages considered were the formulation of research questions, the choice of data sources, search strategies, inclusion and exclusion criteria and quality assessment, and finally, data extraction and synthesis. The results obtained have shown that the use of AR in the classroom provides higher levels of motivation, reflected by factors such as attention, relevance, confidence, and satisfaction, and reflects better results in the tests carried out on the experimental groups compared to the control groups, which means an improvement in the academic performance of students. These results supply a fundamental theoretical basis, where the different teachers should be supported for the incorporation of AR in the classroom, since how this educational technology has been shown offers great opportunities. Likewise, the development of research in areas not so addressed can further clarify the generality of AR based on its influence on learning. In addition, the fields of natural sciences and logical-mathematical have been the most addressed, managing to implement their contents through object modeling. In short, this research highlights the importance of incorporating Augmented Reality into all areas and educational stages, since it is a significant improvement in the teaching and learning process.

Antisense Oligonucleotide-Mediated Reduction of HDAC6 Does Not Reduce Tau Pathology in P301S Tau Transgenic Mice.

Acetylation of tau protein is dysregulated in Alzheimer's Disease (AD). It has been proposed that acetylation of specific sites in the KXGS motif of tau can regulate phosphorylation of nearby residues and reduce the propensity of tau to aggregate. Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme involved in deacetylation of multiple targets, including tau, and it has been suggested that inhibition of HDAC6 would increase tau acetylation at the KXGS motifs and thus may present a viable therapeutic approach to treat AD. To directly test the contribution of HDAC6 to tau pathology, we intracerebroventricularly injected an antisense oligonucleotide (ASO) directed against HDAC6 mRNA into brains of P301S tau mice (PS19 model), which resulted in a 70% knockdown of HDAC6 protein in the brain. Despite a robust decrease in levels of HDAC6, no increase in tau acetylation was observed. Additionally, no change of tau phosphorylation or tau aggregation was detected upon the knockdown of HDAC6. We conclude that HDAC6 does not impact tau pathology in PS19 mice.

Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance.

Huntington's disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-ß lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.

Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington's disease.

A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD(140Q/140Q) mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD(140Q/140Q) mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD(140Q/140Q) neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD(140Q/140Q) neurons. HD(140Q/140Q) mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD(140Q/140Q) neurons.

Striatal synaptosomes from Hdh140Q/140Q knock-in mice have altered protein levels, novel sites of methionine oxidation, and excess glutamate release after stimulation.

BACKGROUND: Synaptic connections are disrupted in patients with Huntington's disease (HD). Synaptosomes from postmortem brain are ideal for synaptic function studies because they are enriched in pre- and post-synaptic proteins important in vesicle fusion, vesicle release, and neurotransmitter receptor activation. OBJECTIVE: To examine striatal synaptosomes from 3, 6 and 12 month old WT and Hdh140Q/140Q knock-in mice for levels of synaptic proteins, methionine oxidation, and glutamate release. METHODS: We used Western blot analysis, glutamate release assays, and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Striatal synaptosomes of 6 month old Hdh140Q/140Q mice had less DARPP32, syntaxin 1 and calmodulin compared to WT. Striatal synaptosomes of 12 month old Hdh140Q/140Q mice had lower levels of DARPP32, alpha actinin, HAP40, Na+/K+-ATPase, PSD95, SNAP-25, TrkA and VAMP1, VGlut1 and VGlut2, increased levels of VAMP2, and modifications in actin and calmodulin compared to WT. More glutamate released from vesicles of depolarized striatal synaptosomes of 6 month old Hdh140Q/140Q than from age matched WT mice but there was no difference in glutamate release in synaptosomes of 3 and 12 month old WT and Hdh140Q/140Q mice. LC-MS/MS of 6 month old Hdh140Q/140Q mice striatal synaptosomes revealed that about 4% of total proteins detected (>600 detected) had novel sites of methionine oxidation including proteins involved with vesicle fusion, trafficking, and neurotransmitter function (synaptophysin, synapsin 2, syntaxin 1, calmodulin, cytoplasmic actin 2, neurofilament, and tubulin). Altered protein levels and novel methionine oxidations were also seen in cortical synaptosomes of 12 month old Hdh140Q/140Q mice. CONCLUSIONS: Findings provide support for early synaptic dysfunction in Hdh140Q/140Q knock-in mice arising from altered protein levels, oxidative damage, and impaired glutamate neurotransmission and suggest that study of synaptosomes could be of value for evaluating HD therapies.

Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington's disease mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD(140Q/140Q)). Primary HD(140Q/140Q) cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD(140Q/140Q) neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD(140Q/140Q) neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

Multiple phenotypes in Huntington disease mouse neural stem cells.

Neural stem (NS) cells are a limitless resource, and thus superior to primary neurons for drug discovery provided they exhibit appropriate disease phenotypes. Here we established NS cells for cellular studies of Huntington's disease (HD). HD is a heritable neurodegenerative disease caused by a mutation resulting in an increased number of glutamines (Q) within a polyglutamine tract in Huntingtin (Htt). NS cells were isolated from embryonic wild-type (Htt(7Q/7Q)) and "knock-in" HD (Htt(140Q/140Q)) mice expressing full-length endogenous normal or mutant Htt. NS cells were also developed from mouse embryonic stem cells that were devoid of Htt (Htt(-/-)), or knock-in cells containing human exon1 with an N-terminal FLAG epitope tag and with 7Q or 140Q inserted into one of the mouse alleles (Htt(F7Q/7Q) and Htt(F140Q/7Q)). Compared to Htt(7Q/7Q) NS cells, HD Htt(140Q/140Q) NS cells showed significantly reduced levels of cholesterol, increased levels of reactive oxygen species (ROS), and impaired motility. The heterozygous Htt(F140Q/7Q) NS cells had increased ROS and decreased motility compared to Htt(F7Q/7Q). These phenotypes of HD NS cells replicate those seen in HD patients or in primary cell or in vivo models of HD. Huntingtin "knock-out" NS cells (Htt(-/-)) also had impaired motility, but in contrast to HD cells had increased cholesterol. In addition, Htt(140Q/140Q) NS cells had higher phospho-AKT/AKT ratios than Htt(7Q/7Q) NS cells in resting conditions and after BDNF stimulation, suggesting mutant htt affects AKT dependent growth factor signaling. Upon differentiation, the Htt(7Q/7Q) and Htt(140Q/140Q) generated numerous Beta(III)-Tubulin- and GABA-positive neurons; however, after 15 days the cellular architecture of the differentiated Htt(140Q/140Q) cultures changed compared to Htt(7Q/7Q) cultures and included a marked increase of GFAP-positive cells. Our findings suggest that NS cells expressing endogenous mutant Htt will be useful for study of mechanisms of HD and drug discovery.

Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer.

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.

Reagents that block neuronal death from Huntington's disease also curb oxidative stress.

Patients with Huntington's disease suffer severe neuronal loss and signs of oxidative damage in the brain. Previously we found that primary neurons from embryonic cortex of mice bearing the Huntington's disease mutation (140 glutamines inserted into exon 1 of huntingtin) showed higher levels of reactive oxygen species before cell death. Here, we treated mutant neurons with known neuroprotective agents and determined the effects on neuronal survival and levels of reactive oxygen species. Primary neurons were exposed to the neurotrophin, brain derived neurotrophic factor, the antioxidant N-acetyl-cysteine or a specific inhibitor of glycogen synthase kinase 3-ß, SB216763. Each reagent increased the survival of the mutant neurons compared with untreated mutant neurons and also reduced the levels of reactive oxygen species to levels of wild-type neurons. These results suggest that reducing the levels of reactive oxygen species may be necessary to protect neurons with the Huntington's disease mutation from cell death.

Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors.

BACKGROUND: The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. RESULTS: Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. CONCLUSION: We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.

Aberrant Rab11-dependent trafficking of the neuronal glutamate transporter EAAC1 causes oxidative stress and cell death in Huntington's disease.

Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD(140Q/140Q)), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD(140Q/140Q) mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.

Mutant huntingtin and glycogen synthase kinase 3-beta accumulate in neuronal lipid rafts of a presymptomatic knock-in mouse model of Huntington's disease.

Patients with Huntington's disease have an expanded polyglutamine tract in huntingtin and suffer severe brain atrophy and neurodegeneration. Because membrane dysfunction can occur in Huntington's disease, we addressed whether mutant huntingtin in brain and primary neurons is present in lipid rafts, which are cholesterol-enriched membrane domains that mediate growth and survival signals. Biochemical analysis of detergent-resistant membranes from brains and primary neurons of wild-type and presymptomatic Huntington's disease knock-in mice showed that wild-type and mutant huntingtin were recovered in lipid raft-enriched detergent-resistant membranes. The association with lipid rafts was stronger for mutant huntingtin than wild-type huntingtin. Lipid rafts extracted from Huntington's disease mice had normal levels of lipid raft markers (G(alphaq), Ras, and flotillin) but significantly more glycogen synthase kinase 3-beta. Increases in glycogen synthase kinase 3-beta have been associated with apoptotic cell death. Treating Huntington's disease primary neurons with inhibitors of glycogen synthase kinase 3-beta reduced neuronal death. We speculate that accumulation of mutant huntingtin and glycogen synthase kinase 3-beta in lipid rafts of presymptomatic Huntington's disease mouse neurons contributes to neurodegeneration in Huntington's disease.

Mutant huntingtin impairs vesicle formation from recycling endosomes by interfering with Rab11 activity.

Huntingtin (Htt) localizes to endosomes, but its role in the endocytic pathway is not established. Recently, we found that Htt is important for the activation of Rab11, a GTPase involved in endosomal recycling. Here we studied fibroblasts of healthy individuals and patients with Huntington's disease (HD), which is a movement disorder caused by polyglutamine expansion in Htt. The formation of endocytic vesicles containing transferrin at plasma membranes was the same in control and HD patient fibroblasts. However, HD fibroblasts were delayed in recycling biotin-transferrin back to the plasma membrane. Membranes of HD fibroblasts supported less nucleotide exchange on Rab11 than did control membranes. Rab11-positive vesicular and tubular structures in HD fibroblasts were abnormally large, suggesting that they were impaired in forming vesicles. We used total internal reflection fluorescence imaging of living fibroblasts to monitor fluorescence-labeled transferrin-carrying transport intermediates that emerged from recycling endosomes. HD fibroblasts had fewer small vesicles and more large vesicles and long tubules than did control fibroblasts. Dominant active Rab11 expressed in HD fibroblasts normalized the recycling of biotin-transferrin. We propose a novel mechanism for cellular dysfunction by the HD mutation arising from the inhibition of Rab11 activity and a deficit in vesicle formation at recycling endosomes.

Disruption of Rab11 activity in a knock-in mouse model of Huntington's disease.

The Huntington's disease (HD) mutation causes polyglutamine expansion in huntingtin (Htt) and neurodegeneration. Htt interacts with a complex containing Rab11GDP and is involved in activation of Rab11, which functions in endosomal recycling and neurite growth and long-term potentiation. Like other Rab proteins, Rab11GDP undergoes nucleotide exchange to Rab11GTP for its activation. Here we show that striatal membranes of HD(140Q/140Q) knock-in mice are impaired in supporting conversion of Rab11GDP to Rab11GTP. Dominant negative Rab11 expressed in the striatum and cortex of normal mice caused neuropathology and motor dysfunction, suggesting that a deficiency in Rab11 activity is pathogenic in vivo. Primary cortical neurons from HD(140Q/140Q) mice were delayed in recycling transferrin receptors back to the plasma membrane. Partial rescue from glutamate-induced cell death occurred in HD neurons expressing dominant active Rab11. We propose a novel mechanism of HD pathogenesis arising from diminished Rab11 activity at recycling endosomes.

Polyglutamine expansion in huntingtin alters its interaction with phospholipids.

Huntingtin has an expanded polyglutamine tract in patients with Huntington's disease. Huntingtin localizes to intracellular and plasma membranes but the function of huntingtin at membranes is unknown. Previously we reported that exogenously expressed huntingtin bound pure phospholipids using protein-lipid overlays. Here we show that endogenous huntingtin from normal (Hdh(7Q/7Q)) mouse brain and mutant huntingtin from Huntington's disease (Hdh(140Q/140Q)) mouse brain bound to large unilamellar vesicles containing phosphoinositol (PI) PI 3,4-bisphosphate, PI 3,5-bisphosphate, and PI 3,4,5-triphosphate [PI(3,4,5)P3]. Huntingtin interactions with multivalent phospholipids were similar to those of dynamin. Mutant huntingtin associated more with phosphatidylethanolamine and PI(3,4,5)P3 than did wild-type huntingtin, and associated with other phospholipids not recognized by wild-type huntingtin. Wild-type and mutant huntingtin also bound to large unilamellar vesicles containing cardiolipin, a phospholipid specific to mitochondrial membranes. Maximal huntingtin-phospholipid association required inclusion of huntingtin amino acids 171-287. Endogenous huntingtin recruited to the plasma membrane in cells that incorporated exogenous PI 3,4-bisphosphate and PI(3,4,5)P3 or were stimulated by platelet-derived growth factor or insulin growth factor 1, which both activate PI 3-kinase. These data suggest that huntingtin interacts with membranes through specific phospholipid associations and that mutant huntingtin may disrupt membrane trafficking and signaling at membranes.

A function of huntingtin in guanine nucleotide exchange on Rab11.

Huntingtin is ubiquitously expressed and enriched in the brain. Deletion of the huntingtin gene in mice is lethal during early embryonic development. The function of huntingtin is, however, not clear. Here, we report that huntingtin is important for the function of Rab11, a critical GTPase in regulating membrane traffic from recycling endosomes to the plasma membrane. In huntingtin-null embryonic stem cells, the levels of Rab11 on membranes and nucleotide exchange activity on Rab11 were significantly reduced compared with normal embryonic stem cells. In brain membranes, an antibody against huntingtin immunoprecipitated a nucleotide exchange activity on Rab11 and huntingtin was coprecipitated with Rab11 in the presence of guanosine diphosphate. These data suggest a role for huntingtin in a complex that activates Rab11.

Role of NADPH oxidase in the apoptotic death of cultured cerebellar granule neurons.

Cerebellar granule neurons (CGN) cultured in a medium containing 25 mM KCl and treated with staurosporine (ST) or transferred to a medium with 5 mM KCl (K5) die apoptotically. CGN death is mediated by an increase in reactive oxygen species (ROS) production. When CGN are treated with antioxidants all apoptotic parameters and cell death are markedly diminished, showing a central role for ROS in this process. Recently, it has been suggested that a possible ROS source involved in cell death is a NADPH oxidase. In that regard, we found expression in CGN of the components of NADPH proteins, p40phox, p47phox and p67phox, and p22phox, as well as three homologues of the catalytic subunit of this complex, NOX1, 2, and 4. The inhibition of NADPH oxidase with diphenylene iodonium or 4-(2-aminoethyl)benzenesulfonyl fluoride significantly reduced ROS production, NADPH oxidase activity, all the apoptotic events, and cell death induced by both K5 and ST. We conclude that ROS could be an early signal of apoptotic neuronal death and that NADPH oxidase, including NOX1, 2, and/or 4, could have a central role in apoptotic death induced by different conditions in these neurons.

Nox1-based NADPH oxidase is the major source of UVA-induced reactive oxygen species in human keratinocytes.

UVA radiation is a major environmental stress on skin, causing acute and chronic photodamage. These responses are mediated by reactive oxygen species (ROS), although the cellular source of these ROS is unknown. We tested the hypotheses that UVA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required for ROS generation in human keratinocytes (HK) and that these ROS initiate rapid prostaglandin E2 (PGE2) synthesis. Treatment of HK with a non-toxic dose of UVA rapidly increased NADPH oxidase activity and intracellular ROS, which were partially blocked by an inhibitor of NADPH oxidase and by a mitochondria-selective antioxidant. Depleting the Nox1 isoform of the catalytic subunit of NADPH oxidase using small interfering RNA (siRNA) blocked the UVA-induced ROS increase, indicating that ROS produced by mitochondria or other sources are downstream from Nox1. Nox1 siRNA also blocked UVA-initiated PGE2 synthesis. The mechanism for activation of Nox1 is mediated by an increase in intracellular calcium. Ceramide, which has been proposed to mediate responses to UVA in HK, also activated NADPH oxidase. These results indicate that UVA activates Nox1-based NADPH oxidase to produce ROS that stimulate PGE2 synthesis, and that Nox1 may be an appropriate target for agents designed to block UVA-induced skin injury.

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts.

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.