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.
KeywordsAutophagy Protein aggregation Sequestosome-1 p62 Cortex Ependyma Choroid plexus
Autophagy is a mechanism for the delivery of cell components to lysosomes for hydrolytic degradation. The main type of autophagy is macroautophagy which involves the formation of double-membraned vesicles, known as autophagosomes, around substrates. A set of autophagy-related genes, such as Atg5 and Atg7, is essential for this process, and deletion of these genes suppresses autophagy in mice [1, 2]. Adaptor proteins such as sequestosome 1, also known as p62 , differentially bind to autophagy substrates and introduce specificity into the degradation process . Autophagy removes many types of protein aggregates, dysfunctional organelles, and other potentially dangerous cell components but also contributes to the recycling of macromolecules to ensure cellular homeostasis [3, 5, 6, 7, 8].
Sequestosome-1/p62 is a multifunctional protein comprising domains that bind to the mammalian Atg8 homolog microtubule-associated protein 1 light chain 3 (LC3), which mediates docking of autophagy substrates to the forming autophagosome, ubiquitinated proteins, and the Nrf2 regulator Keap1 . Via its N-terminal Phox and Bem1p (PB1) domain, p62 is able to self-oligomerize in the form of filaments . Suppression of autophagy results in the intracellular accumulation of p62 . p62 is present in neurofibrillary tangles in Alzheimer’s disease and Lewy bodies in Parkinson’s disease . Together with reports about the decline of autophagic activity in aged organs and impaired clearance of autophagosomes in neurodegenerative diseases, aberrant processing of p62 in diseased tissues has suggested a particularly important role of autophagy in the aging brain [10, 11, 12]. Accordingly, the pharmacological inducer of autophagy, rapamycin, has been suggested as therapeutic agent for aging-associated neurodegeneration [13, 14].
Cell types of different functions and turnover rates vary in their dependence on autophagy for the elimination of damaged organelles and potentially harmful protein aggregates as well as for recycling of building blocks of macromolecules [3, 8, 15]. While constitutive deletion of either Atg5 or Atg7 leads to perinatal lethality in mice [16, 17], cell type-specific deletions of autophagy genes via the Cre-loxP system allows to inactivate autophagy in a targeted manner and to determine whether lack of autophagy plays essential roles in these specific cells [1, 2].
In previous studies, we have generated Atg7f/f Tyr-Cre mice for the investigation of the role of autophagy in pigment cells [18, 19, 20]. The Tyr-Cre gene utilizes promoter and enhancer elements from tyrosinase (Tyr), a gene encoding the enzyme that converts tyrosine to melanin via tyrosine hydroxylase and dopa oxidase catalytic activities in pigment cells. The Tyr promoter drives the expression of a transgene encoding the Cre recombinase, which deletes the region between two loxP sites. The target sites have been introduced into an essential part of the autophagy gene Atg7 , so that the expression of Cre in cells with Tyr promoter activity leads to the permanent inactivation of Atg7. When these cells proliferate, Atg7-dependent autophagy remains suppressed in all progeny cells. Atg7f/f Tyr-Cre mice show mild hypopigmentation of hair and tail skin  but otherwise appear phenotypically normal. Autophagy is also suppressed in the retinal pigment epithelium of Atg7f/f Tyr-Cre mice leading to the accumulation of p62 and an increase in the abundance of a degradation-prone variant of retinal pigment epithelium-specific 65 kDa protein (RPE65) .
The characterization of mice carrying the Tyr-Cre transgene has shown that Cre expression and Cre-mediated gene deletions do not only occur in pigment cells but also in distinct groups of neurons of the developing brain [21, 22]. Specifically, Tyr-Cre-mediated gene deletions were reported in the basal forebrain, hippocampus (dentate gyrus pyramidal cell layers), olfactory bulb, the granule cell layer of the lateral cerebellum cortex, sympathetic cephalic ganglia, leptomeninges of the telencephalon, and cranial nerves (V), (VII), and (IX) . By contrast, the neuroepithelial cells of the adult brain such as the ependyma and the choroid plexus epithelium have not been reported to be affected by Tyr-Cre-mediated DNA recombination [21, 22].
Here we investigated Atg7f/f Tyr-Cre mice for p62 accumulations signifying suppression of autophagy in non-pigment cells. We show that p62 accumulates in neuroepithelial cells of the ocular ciliary body, the choroid plexus and the ependyma as well as in neurons of the brain. By immunogold labeling and electron microscopy, the ultrastructure of these p62 aggregates is revealed to consist of filaments both in neurons and neuroepithelial cells. Our data establish Atg7f/f Tyr-Cre mice as a model for the study of aging-associated aberrant p62 depositions in cells of the neuroectodermal lineage.
Material and Methods
The generation and maintenance of Atg7f/f Tyr-Cre mice have been reported previously . Briefly, Atg7f/f mice (kindly provided by Masaaki Komatsu, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) were crossed with mice carrying the Tyr-Cre transgene . Tissue samples were prepared from age-matched Atg7f/f Tyr-Cre and Atg7f/f mice. Only hemizygous males and homozygous females were included to avoid possible effects of X chromosome inactivation on the Tyr-Cre transgene in heterozygous females .
Immunohistochemical and Immunofluorescence Analysis
For histological investigations, the eyes were enucleated immediately after sacrificing mice. Likewise, the brain and other tissues were prepared. The tissue samples were fixed in 4% paraformaldehyde over night and then embedded in paraffin. Thin-sections were investigated by immunohistochemistry and immunofluorescence labelling according to published protocols  with modifications. The sections were incubated with polyclonal rabbit anti-Sqstm1/p62 (MBL International Corporation, dilution, 1:1000) followed by incubation with goat anti-rabbit immunoglobulin conjugated to horseraddish peroxidase for 30 min. In immunofluorescence double labelings, anti-p62 was used besides mouse monoclonal anti-tyrosine hydroxylase (Millipore, MAB318, clone LNC1, 1: 400) and mouse monoclonal anti-ubiquitin (Millipore, 1:500). The following secondary antibodies were used for immunofluorescence labeling: goat anti-rabbit immunoglobulin coupled to Alexa-Fluor 488 (green) or Alexa-Fluor 546 (red) (Molecular Probes, Leiden, The Netherlands), and goat anti-mouse immunoglobulin coupled to Alexa Fluor 546 (Life Technology, 1:500). Counterstaining of nuclei was done with hematoxylin for immunohistochemistry and Hoechst 33258 (Molecular Probes) for immunofluorescence analysis. Isotype antibodies of unrelated specificities were used instead of the primary antibodies in negative control experiments. The labeled sections were photographed under a fluorescence microscope using the Metamorph software.
Immunogold Labeling and Electron Microscopy
Whole brains were immersely fixed in immune fixative containing 3.2% paraformaldehyde, 0.2% glutaraldehyde, 1% sucrose, and 3 mM CaCl2 in 0.1 M Na-cacodylate buffer for overnight incubation at 4 °C. Pieces of 3 × 3 × 3 mm of the lateral ventricle wall were resected. The small tissue blocks were cryoprotected in 30% sucrose in Na-cacodylate for 24 h. The blocks were frozen in liquid nitrogen and subsequently transferred to anhydrous methanol containing 0.5% uranyl-acetate at − 70 °C. After 6 h, the temperature was raised to − 20 °C and the dehydration was continued for 24 h with gentle agitation. Then specimens were infiltrated with pure LR Gold at − 20 °C for 24 h (three incubations of 8 h each) and then polymerized for 96 h at − 20 °C using a DL-103 12 W ultraviolet lamp.
Ultrathin sections were collected on formvar film-coated nickel grids. For epitope retrieval and quenching, the samples were treated with 0.3% Na-borohydride in Tris-buffered saline containing 50 mM NH4Cl and 50 mM glycine for 10 min at room temperature. After antigen retrieval, the samples were incubated with affinity-purified rabbit polyclonal anti-p62 antibody (1:100 dilution, overnight at 4 °C), followed by incubation with goat anti-rabbit immunoglobuin secondary antibody conjugated with 10 nm gold particles (Sigma Aldrich, 1:100, 6 h at room temperature). The immuno-labeled sections were counterstained with uranyl acetate and lead citrate prior to investigation with a JEOL JEM-1011 electron microscope.
Western Blot Analysis
Brains were lysed in a protein extraction buffer containing 50 mM Tris (pH 7.4), 2% SDS, and complete protease inhibitor cocktail (Roche, Mannheim, Germany) and homogenized by sonication. The insoluble debris was removed by centrifugation, and the protein concentration of the supernatant was measured by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Western blot analysis was performed as described previously . Twenty microgram protein was loaded per lane on SDS polyacrylamide gels (ExcelGel SDS, gradient 8–18, Amersham Biosciences) on a horizontal electrophoresis system (Amersham Biosciences) and thereafter blotted onto a nitrocellulose membrane. For the detection of specific antigens, the following first step antibodies were used: rabbit polyclonal anti-p62 (BML-PW9860-0100, Enzo Life Sciences, NY, dilution 1:2000), rabbit anti-Atg7 (Sigma, 1:1000), and mouse anti-GAPDH (HyTest Ltd., Finland, 1:2000). As secondary antibodies, goat anti-rabbit immunoglobulin G (IgG) (Bio-Rad Laboratories, CA) and sheep anti-mouse immunoglobulin G (GE Healthcare, UK) antibodies conjugated to horseradish peroxidase were used at a dilution of 1:10000. The bands were revealed with enhanced chemiluminescence reagent (ThermoFisher Scientific).
RNA Preparation, Reverse Transcription, and Quantitative PCRs
RNA was prepared from brains using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocol. Quantitative real-time PCRs with SYBR-Green in the LightCycler system (Roche Applied Science, Mannheim, Germany) were performed according to a published protocol [18, 19]. Transcripts of the following genes were amplified with the indicated primers: Beta-2 microglobulin (B2m) (Mm_B2m_f, 5′-attcacccccactgagactg-3′ and Mm_B2m_r, 5′-tgctatttctttctgcgtgc-3′), γ-glutamyl cystine ligase modulatory subunit (Gclm) (Mm_Gclm_f, 5′-tggagcagctgtatcagtgg-3′ and Mm_Gclm_r, 5′-agagcagttctttcgggtca-3′), NAD(P)H:quinone oxidoreductase 1 (Nqo1) (Mm_Nqo1_f, 5′-gaagctgcagacctggtgat-3′ and Mm_Nqo1_r, 5′-ttctggaaaggaccgttgtc-3′), and Sqstm-1/p62 (Mm_p62_f, 5′-ccagtgatgaggagctgaca-3′ and Mm_p62_r, 5′-tgggcacacactgcacttat-3′) .
Preparation and Quantification of Lipids
Mouse brain tissue (n = 4 per genotype) was homogenized in the ninefold volume of methanol/acetic acid (3%)/butylated hydroxytoluene (BHT, as antioxidant, 0.01%). Samples were purified using the liquid-liquid extraction procedure  and were reconstituted in 85% aqueous methanol containing 5 mM ammonium formate and 0.1% formic acid. Analysis was performed at FTC-Forensic Toxicological Laboratory, Vienna. Aliquots (10 μl) were injected onto a core-shell type C 18 column (Kinetex 2.6 μm, 50 mm × 3.0 mm ID; Phenomenex, Torrance, CA) kept at 20 °C and using a 1200 series HPLC system (Agilent, Waldbronn, Germany), coupled to a 4000 QTrap triple quadrupole linear ion trap hybrid mass spectrometer system with a Turbo V electrospray ion source (Applied Biosystems, Foster City, CA, USA). Elution was performed according to a published protocol . Detection was carried out in positive ion mode by selected reaction monitoring (SRM) of 99 MS/MS transitions using product ion (m/z 184), the diagnostic fragment for the phosphocholine residue. Data acquisition and instrument control were performed with Analyst software, version 1.6 (Applied Biosystems). Individual values were normalized to the intrinsic 1,2-di-palmitoyl-3-phosphorylcholine (DPPC) for brain extracts. Non-oxidized native lipid species (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) m/z 782; 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphorylcholine (PLPC) m/z 758; 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC) m/z 810; 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphorylcholine (SLPC) m/z 786) and chain fragmented oxidized species (1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) m/z 594; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphorylcholine (PAzPC) m/z 666; 1-stearoyl-2-azelaoyl-sn-glycero-3-phosphorylcholine (SAzPC) m/z 694 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC) m/z 610; 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycero-3-phosphorylcholine (PONPC) m/z 650 and 1-stearoyl-2-(oxo-nonanoyl)-sn-glycero-3-phosphorylcholine (SONPC) m/z 678) were identified as isobaric and co-eluting with commercial standards .
Preparation of CSF and Analysis of Proteins by Electrophoresis
The statistical significance of differences between sample groups was examined using the two-tailed unpaired Student’s t test. P values below 0.05 were considered significant.
Mice were maintained and sacrificed by cervical dislocation according to the animal welfare guidelines of the Medical University of Vienna, Austria, as approved by the Ethics Review Committee for Animal Experimentation of the Medical University of Vienna, Austria, and the Federal Ministry of Science, Research and Economy, Austria (GZ 66.009/0255-II/3b/2013). CSF was prepared under approval of the Federal Ministry of Science, Research and Economy, Austria (BMWFW-66.010/0045-WF/V/3b/2015). All methods were performed in accordance with the relevant guidelines and regulations.
Tyr-Cre-Mediated Deletion of Atg7 Leads to Accumulation of p62 in Neurons and Neuroepithelial Cells of the Brain
As the Tyr-Cre transgene has been reported to be active not only in pigment cells but also in other cells of the neuroectodermal lineage [21, 22], we analyzed the brain of Atg7f/f Tyr-Cre mice and, as control, the brain of fully autophagy-competent Atg7f/f mice. Both male and female Atg7f/f Tyr-Cre mice were successfully used in breeding, had normal weight, and could be kept up to an age of 2 years. However, Atg7f/f Tyr-Cre mice older than 1.5 years showed abnormal limb-clasping reflexes that were also reported in mouse models of neurodegenerative diseases [1, 2, 28] (Suppl. Fig. S1). To determine possible changes in the central nervous system of Atg7f/f Tyr-Cre mice, brains of young (age 1–2 months) and old (age 14–27 months) mice of both genotypes (Atg7f/f Tyr-Cre and Atg7f/f mice) were investigated.
Atg7 f/f Tyr-Cre-Induced Aggregates of p62 Are Not Strictly Associated with Ubiquitin
Immuno-labeling for ubiquitin showed that some but not all p62 bodies in the brains of Atg7f/f Tyr-Cre mice were associated with immunoreactivity for ubiquitin (Fig. 3). Only in few neurons, mainly located in the cortex (Fig. 3J–L), the relative increase of ubiquitin was as pronounced as that observed for p62, whereas many cells with p62 accumulations did not have increased levels of ubiquitin.
Electrophoretic analysis suggested that the cerebrospinal fluid (CSF), which is secreted by the choroid plexus epithelium and, to a smaller extent, by the ependyma , contained the same major protein species at the same relative abundance in Atg7f/f Tyr-Cre and Atg7f/f mice (Suppl. Fig. S5). Thus, Tyr-Cre-mediated deletion of Atg7 caused aberrant accumulation of the autophagy substrate p62 without deleterious effects on the secretory function of neuroepithelial cells.
Immunogold Electron Microscopy Shows Filamentous Structure of p62 Aggregates in Epithelial Cells of the Ependyma and Neurons of Atg7 f/f Tyr-Cre Mice
Tyr-Cre-Mediated Deletion of Atg7 Is Associated with an Increase in Dicarboxylic Acid-Containing Phospholipids in the Brain
p62 Accumulates in the Ciliary Body of the Eye of Atg7 f/f Tyr-Cre Mice
The results of this study establish Atg7f/f Tyr-Cre mice as a model for the study of autophagy-deficiency in non-pigment cells of the neuroectodermal lineage and support the hypothesis of filament formation of endogenous p62 as a mechanism of sequestering p62 and possibly other proteins in autophagy-deficient cells. Given the emerging role of p62 as a multi-functional adapter protein [4, 33], our findings are likely relevant for several aspects of neurobiology.
In the present study, the Tyr-Cre transgene [21, 23] was used to delete the floxed alleles of Atg7. The transgenic tyrosinase promoter was shown to be active in some neural crest cell precursors of melanocytes, some smooth muscle cells of the heart and cells of the enteric nervous system, but also in the brain [21, 23, 34, 35]. The neuronal expression of Tyr-Cre was used to delete phosphatase and tensin homolog (Pten) in a subset of vagal neural crest cells, resulting in lethal intestinal pseudoobstruction of Tyr-Cre/° Ptenf/f mice . We have previously investigated the effects of Tyr-Cre mediated deletion of Atg7 on skin melanocytes and retinal pigment epithelial cells [18, 20]. Atg7f/f Tyr-Cre mice displayed mild defects in hair pigmentation and alteration in the turnover of the C57BL/6 background-specific M450 variant of RPE65 and reached an age beyond 2 years. Of note, tyrosinase is not involved in the synthesis of neuromelanin, the pigment within the substantia nigra, a region of the midbrain , and accordingly, a specific deletion of Atg7 was not expected in the substantia nigra of Atg7f/f Tyr-Cre mice. However, in line with the reported expression of the Tyr-Cre transgene in multiple non-pigmented neuroectodermal cells, we found a diverse set of phenotypically abnormal cells. The present characterization of p62 aggregations in a series of neuroectodermal lineage cells of Atg7f/f Tyr-Cre mice is an important extension of previous studies because it suggests that cell-autonomous effects, such as those on melanocytes, may be accompanied by effects of altered signaling from neurons in Atg7f/f Tyr-Cre mice.
The accumulation of p62 in neurons and neuroepithelial cells of the brain and the eyes of Atg7f/f Tyr-Cre mice indicates that Atg7-dependent autophagy is abrogated in these cells. The reduced degradation of p62 is an accepted in vivo marker of impaired autophagy [38, 39], especially when supported by evidence for lack of transcriptional upregulation of Sqstm1/p62 expression (Suppl. Fig. S4). Importantly, the subcellular abnormalities in the brain of Atg7f/f Tyr-Cre mice are associated with abnormal hindlimb clasping reflexes at an age of 1.5 years and more in Atg7f/f Tyr-Cre mice (Suppl. Fig. S1) but do not impair the survival of mice and therefore differ from the effects of Atg7 and Atg5 deletions in all neurons which are lethal within 4–28 weeks after birth of mice [1, 2]. Thus, Atg7f/f Tyr-Cre mice extend the list of viable mouse lines carrying deletions of Atg7 in distinct sets of brain neurons [40, 41, 42, 43, 44, 45, 46, 47, 48, 49]. Accordingly, Atg7f/f Tyr-Cre mice will be available for studying the impact of lack of autophagy and aberrant accumulation of p62 in neurons of the brain in future studies.
Unexpectedly, neuroepithelial cells of the choroid plexus, the ependyma, and the ocular ciliary body were also affected by Tyr-Cre-driven inactivation of Atg7. To the best of our knowledge, conditional suppression of autophagy has not been reported previously in these cell types. The structure of p62 bodies in ependymal cells was similar to that in neurons, and the p62 aggregates in the ciliary body were even larger than those in neurons. The neuroepithelia of the choroid plexus and the ciliary body do not only share common embryological origins ; they also have similar functions. Both of these epithelia secrete liquids, namely the cerebrospinal fluid and the aqueous humor of the eye, respectively. As the secretion rates of these epithelia control the intracranial pressure and the intraocular pressure, defects of the choroid plexus epithelium and the ciliary body epithelium may be medically relevant. It remains to be investigated whether Tyr-Cre-driven gene recombination can be used to study functional parameters pertaining to the etiology of hydrocephalus or glaucoma. Choroid plexus epithelial cells of Atg7f/f Tyr-Cre mice developed p62 bodies predominantly in the cell periphery, reminiscent of the so-called Biondi bodies or Biondi ring tangles that appear during human aging and increase significantly in patients with Alzheimer’s disease [51, 52]. Although the structures of the p62 aggregates in the mutant mice and those of the tangles in humans are different , possible similarities in mechanisms of formation should be investigated.
Our data suggest that p62 forms filamentous aggregates when sufficiently high-intracellular concentrations are reached due to lack of autophagic turnover. In Atg7f/f Tyr-Cre mice, p62 was diffusely distributed in melanocytes  and some brain neurons (Fig. 1) whereas in most of the affected neurons and neuroepithelial cells, p62 was concentrated in aggregates, also referred to as p62 bodies. The formation of p62 bodies has also been reported for other mouse models of autophagy deficiency and therefore appears to be a characteristic feature of p62 . Our investigation of the ultrastructural organization of p62 bodies demonstrates filaments of uniform thickness that were densely bound by anti-p62 antibodies. Previous studies have suggested that protein aggregates containing endogenous p62 have a filamentous organization, e.g., in dendrites of dopaminergic neurons of mice that carry a tyrosine hydroxylase (TH) cell-specific deletion of Atg7 (Atg7f/f;TH-IRES-Cre) . However, only recently evidence from studies involving recombinant p62 in vitro [55, 56] and recombinant p62 expressed by an adeno-associated virus vector injected into the rat substantia nigra  have suggested that p62 itself is sufficient to form filaments. The structure and the dimensions of the p62 filaments in neurons and neuroepithelial cells of Atg7f/f Tyr-Cre mice is similar or identical to those reported for pure p62  indicating that endogenous p62 is the main, if not the only, component of these filaments. Our double-immunofluorescence labeling results suggest that ubiquitin is also present at elevated concentrations in p62 bodies of Atg7f/f Tyr-Cre mice; however, the relative abundance inside versus outside of these aggregates was much lower for ubiquitin than p62. Furthermore, besides ubiquitin-positive aggregates, apparently ubiquitin-negative aggregates were detected, suggesting that ubiquitinated proteins do not play an essential role in p62 body formation. Thus, we propose that the formation of p62 bodies in Atg7f/f Tyr-Cre mice is likely driven by an inherent tendency to filamentous polymerization of p62 at elevated concentrations.
Our demonstration of elevated amounts of the oxidized phospholipids PAzPC and SAzPC in Atg7f/f Tyr-Cre brains indicates that the turnover of these substances is altered. The differential effects of autophagy inhibition on different classes of oxidized phospholipids (PAzPC and SAzPC versus Lyso-PPC, Lyso-SPC, PLPC, and SLPC in the Atg7f/f Tyr-Cre brain), which are similar to those observed in Atg7f/f K14-Cre skin cells , may be caused by the delivery of substrates to lysosomal phospholipase A2 . Augmentation of oxidatively modified phospholipids in cellular membranes affects their polarity and permeability, which has been specifically demonstrated for PLPC-derived PazPC . Additionally, PAzPC has been identified as a chemical modifier of proteins in oxidative stress , and as inducer of amyloid fibril aggregation . Further research will address possible mechanistic links between phospholipid oxidation and the protein aggregates in the brain.
In summary, our results suggest that Tyr-Cre-mediated suppression of Atg7-dependent autophagy causes the aggregation of p62 in neurons and neuroepithelial cells and an increase of oxidized lipid species in the brain. Because of the apparently slow accumulation of damage, Atg7f/f Tyr-Cre mice can be used to study the effects of p62 aggregation in neuroectoderm-derived cells during aging.
Open access funding provided by Medical University of Vienna. This project was supported by the Herzfelder’sche Familienstiftung. We thank Masaaki Komatsu (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for providing Atg7-floxed mice, Johannes Hainfellner and Andreas Pollreisz for helpful discussions, and Ferencne Truszka for the technical assistance in electron microscopy. The financial support of the Federal Ministry for Digital and Economic Affairs (BMDW) of Austria and the National Foundation for Research, Technology, and Development of Austria to the Christian Doppler Laboratory for Biotechnology of Skin Aging is gratefully acknowledged. LE and FG are participants of the COST Action CA15138, European Network of Multidisciplinary Research and Translation of Autophagy knowledge (TRANSAUTOPHAGY) that is supported by the European Union Framework Programme Horizon 2020.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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