Imaging of metabolic activity adaptations to UV stress, drugs and differentiation at cellular resolution in skin and skin equivalents – Implications for oxidative UV damage

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Redox Biology / Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo

Autophagy allows cells fundamental adaptations to metabolic needs and to stress. Using autophagic bulk degradation cells can clear crosslinked macromolecules and damaged organelles that arise under redox stress. Accumulation of such debris results in cellular dysfunction and is observed in aged tissue and senescent cells. Conversely, promising anti-aging strategies aim at inhibiting the mTOR pathway and thereby activating autophagy, to counteract aging associated damage. We have inactivated autophagy related 7 (Atg7), an essential autophagy gene, in murine keratinocytes (KC) and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by paraquat (PQ), an oxidant drug commonly used to induce cellular senescence. Atg7 deficient KC displayed increased prostanoid signaling and a pro- mitotic gene expression signature as compared to the WT. After exposure to PQ, both WT and KO cells showed an inflammatory and stress-related transcriptomic response. However, the Atg7 deficient cells additionally showed drastic DNA damage- and cell cycle arrest signaling. Indeed, DNA fragmentation and oxidation were strongly increased in the stressed Atg7 deficient cells upon PQ stress but also after oxidizing ultraviolet A irradiation. Damage associated phosphorylated histone H2AX (H2AX) foci were increased in the nuclei, whereas expression of the nuclear lamina protein lamin B1 was strongly decreased. Similarly, in both, PQ treated mouse tail skin explants and in UVA irradiated mouse tail skin, we found a strong increase in H2AX positive nuclei within the basal layer of Atg7 deficient epidermis. Atg7 deficiency significantly affected expression of lipid metabolic genes. Therefore we performed lipid profiling of keratinocytes which demonstrated a major dysregulation of cellular lipid metabolism. We found accumulation of autophagy agonisitic free fatty acids, whereas triglyceride levels were strongly decreased. Together, our data show that in absence of Atg7/autophagy the resistance of keratinocytes to intrinsic and environmental oxidative stress was severely impaired and resulted in DNA damage, cell cycle arrest and a disturbed lipid phenotype, all typical for premature cell aging.(VLID)485638

Consequences of Autophagy Deletion on the Age-Related Changes in the Epidermal Lipidome of Mice

Autophagy is a controlled mechanism of intracellular self-digestion with functions in metabolic adaptation to stress, in development, in proteostasis and in maintaining cellular homeostasis in ageing. Deletion of autophagy in epidermal keratinocytes does not prevent the formation of a functional epidermis and the permeability barrier but causes increased susceptibility to damage stress and metabolic alterations and accelerated ageing phenotypes. We here investigated how epidermal autophagy deficiency using Keratin 14 driven Atg7 deletion would affect the lipid composition of the epidermis of young and old mice. Using mass spectrometric lipidomics we found a reduction of age-related accumulation of storage lipids in the epidermis of autophagy-deficient mice, and specific changes in chain length and saturation of fatty acids in several lipid classes. Transcriptomics and immunostaining suggest that these changes are accompanied by changes in expression and localisation of lipid and fatty acid transporter proteins, most notably fatty acid binding protein 5 (FABP5) in autophagy knockouts. Thus, maintaining autophagic activity at an advanced age may be necessary to maintain epidermal lipid homeostasis in mammals

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7f/f Tyr-Cre mice relative to Atg7f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.(VLID)359835

Redox Biology / A novel role for NUPR1 in the keratinocyte stress response to UV oxidized phospholipids

Ultraviolet light is the dominant environmental oxidative skin stressor and a major skin aging factor. We studied which oxidized phospholipid (OxPL) mediators would be generated in primary human keratinocytes (KC) upon exposure to ultraviolet A light (UVA) and investigated the contribution of OxPL to UVA responses. Mass spectrometric analysis immediately or 24h post UV stress revealed significant changes in abundance of 173 and 84 lipid species, respectively. We identified known and novel lipid species including known bioactive and also potentially reactive carbonyl containing species. We found indication for selective metabolism and degradation of selected reactive lipids. Exposure to both UVA and to in vitro UVA - oxidized phospholipids activated, on transcriptome and proteome level, NRF2/antioxidant response signaling, lipid metabolizing enzyme expression and unfolded protein response (UPR) signaling. We identified NUPR1 as an upstream regulator of UVA/OxPL transcriptional stress responses and found this protein to be expressed in the epidermis. Silencing of NUPR1 resulted in augmented expression of antioxidant and lipid detoxification genes and disturbed the cell cycle, making it a potential key factor in skin reactive oxygen species (ROS) responses intimately involved in aging and pathology.(VLID)490413

<i>Tyrosinase-Cre</i>-Mediated Deletion of the Autophagy Gene <i>Atg7</i> Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

<div><p>Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene <i>Atg7</i>. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to <i>Sqstm1</i>, genes involved in retinal regeneration, i.e. <i>Lrat</i>, <i>Rdh5</i>, <i>Rgr</i>, and <i>Rpe65</i>, were expressed at higher mRNA levels. Sequencing of the <i>Rpe65</i> gene showed that <i>Atg7</i>^<i>f/f</i> and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice is, at least partly, mediated by upregulation of <i>Rpe65</i> transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by <i>Atg7</i>-dependent autophagy in <i>Atg7</i>^<i>f/f</i> mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.</p></div

Deletion of Atg7 leads to the accumulation of p62 and RPE65 in the RPE.

<p>Double-immunofluorescence labelling of p62 (green) (A, B) and RPE65 (red) (C, D) in <i>Atg7</i>^<i>f/f</i> (A, C, E) and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> (B, D, F) eyes. Nuclear DNA was labelled with Hoechst 33258 (blue). Panels E and F show merged images. ONL, outer nuclear layer; PRL, photoreceptor layer; RPE, retinal pigment epithelium; Ch, choroid. Scale bars: 50 μm.</p

Regulators of the visual cycle are expressed at increased levels in the RPE of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice.

<p>The expression levels of genes encoding visual cycle regulators (<i>Lrat</i>, <i>Rpe65</i>, <i>Rdh5</i>, <i>Rgr</i>), transcription factors implicated in the homeostasis of the RPE (<i>Sox9</i>, <i>Otx2</i>), and house-keeping genes (<i>Alas1</i>, <i>B2m</i>) were determined by quantitative RT-PCR analysis of RNAs from the RPE of <i>Atg7</i>^<i>f/f</i> (n = 5) and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> (n = 4) mice. Expression levels (a.u., arbitrary units) are shown relative to the expression of the house-keeping gene <i>B2m</i>. Error bars indicate standard deviations. *<i>p</i><0.05, considered statistically significant (two-tailed t-test). n.s., not significant.</p