Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1^flox/flox). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.     

Intracellular Quality Control by Autophagy: How Does Autophagy Prevent Neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5^-/- mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5^flox/flox;Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Aberrant Membranes and Double-Membrane Structures Accumulate in the Axons of Atg5-Null Purkinje Cells before Neuronal Death

Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes (Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5^flox/flox with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell–specific promoter, pcp2. In Atg5^flox/flox; pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons.     

aP2-Cre-Mediated Inactivation of Estrogen Receptor Alpha Causes Hydrometra

In this study we describe the reproductive phenotypes of a novel mouse model in which Cre-mediated deletion of ERα is regulated by the aP2 (fatty acid binding protein 4) promoter. ERα-floxed mice were crossed with transgenic mice expressing Cre-recombinase under the control of the aP2 promoter to generate aP2-Cre/ERα^flox/flox mice. As expected, ERα mRNA levels were reduced in adipose tissue, but in addition we also detected an 80% reduction of ERα levels in the hypothalamus of aP2-Cre/ERα^flox/flox mice. Phenotypic analysis revealed that aP2-Cre/ERα^flox/flox female mice were infertile. In line with this, aP2-Cre/ERα^flox/flox female mice did not cycle and presented 3.8-fold elevated estrogen levels. That elevated estrogen levels were associated with increased estrogen signaling was evidenced by increased mRNA levels of the estrogen-regulated genes lactoferrin and aquaporin 5 in the uterus. Furthermore, aP2-Cre/ERα^flox/flox female mice showed an accumulation of intra-uterine fluid, hydrometra, without overt indications for causative anatomical anomalies. However, the vagina and cervix displayed advanced keratosis with abnormal quantities of accumulating squamous epithelial cells suggesting functional obstruction by keratin plugs. Importantly, treatment of aP2-Cre/ERα^flox/flox mice with the aromatase inhibitor Letrozole caused regression of the hydrometra phenotype linking increased estrogen levels to the observed phenotype. We propose that in aP2-Cre/ERα^flox/flox mice, increased serum estrogen levels cause over-stimulation in the uterus and genital tracts resulting in hydrometra and vaginal obstruction.     

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.     

Specific Disruption of Tsc1 in Ovarian Granulosa Cells Promotes Ovulation and Causes Progressive Accumulation of Corpora Lutea

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor negatively regulating mammalian target of rapamycin complex 1 (mTORC1). It is reported that mice lacking Tsc1 gene in oocytes show depletion of primordial follicles, resulting in premature ovarian failure and subsequent infertility. A recent study indicated that deletion of Tsc1 in somatic cells of the reproductive tract caused infertility of female mice. However, it is not known whether specific disruption of Tsc1 in granulosa cells influences the reproductive activity of female mice. To clarify this problem, we mated Tsc1^flox/flox mice with transgenic mice strain expressing cyp19-cre which exclusively expresses in granulosa cells of the ovary. Our results demonstrated that Tsc1^flox/flox; cyp19-cre mutant mice were fertile, ovulating more oocytes and giving birth to more pups than control Tsc1^flox/flox mice. Progressive accumulation of corpora lutea occurred in the Tsc1^flox/flox; cyp19-cre mutant mice with advanced age. These phenotypes could be explained by the elevated activity of mTORC1, as indicated by increased phosphorylation of rpS6, a substrate of S6 in the Tsc1^flox/flox; cyp19-cre mutant granulosa cells. In addition, rapamycin, a specific mTORC1 inhibitor, effectively rescued the phenotype caused by increased mTORC1 activity in the Tsc1^cko ovaries. Our data suggest that conditional knockout of Tsc1 in granulosa cells promotes reproductive activity in mice.     

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.     

E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13^flox/floxK5-Cre). At birth, the skin of the Ubc13^flox/floxK5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13^flox/floxK5-Cre mouse epidermis. In culture, Ubc13^flox/floxK5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13^flox/flox keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13^flox/floxK5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.     

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch''s membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch''s membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4^−/− and Rdh8^−/−/Abca4^−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4^−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4^−/− mice revealed that carbonyl adduct deposition in Bruch''s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4^−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch''s membrane that can confer risk of age-related macular degeneration.     

Conditional Mesenchymal Disruption of Pkd1 Results in Osteopenia and Polycystic Kidney Disease

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1 ^flox/flox and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1 ^flox/flox and Pkd1 ^flox/+, heterozygous Col1a1(3.6)-Cre;Pkd1 ^flox/+ and Pkd1 ^flox/null, and homozygous Col1a1(3.6)-Cre;Pkd1 ^flox/flox and Col1a1(3.6)-Cre;Pkd1 ^flox/null mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1 ^flox/null mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1 ^flox/+ and Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation.     

Transcription Factor Nrf1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes

Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1^flox allele and a rat CYP1A1-Cre transgene (Nrf1^flox/flox::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1^flox/flox::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1^flox/flox::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1^flox/flox::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x_c⁻, was significantly upregulated in livers of Nrf1^flox/flox::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.     

Autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors

Autophagy has been demonstrated to be associated with the pathogenesis of cancer, but no consensus has been reached about its precise role. Therefore, we investigated whether autophagy in the intestinal epithelium is involved in the pathogenesis of intestinal tumors. To evaluate the relationship between autophagy and intestinal tumors, GFP-LC3-APC(min/+) mice were generated by mating GFP-LC3 transgenic mice with APC(min/+) mice. Autophagy was weakly induced in the intestinal polyp regions of the mice in comparison to their non-polyp regions. Under starved conditions, autophagy was not induced in the polyp regions, whereas it was observed in the non-polyp regions. Then, to examine whether a lack of autophagy in the intestinal epithelium enhances the induction of intestinal tumor, Atg7flox/flox:vil-cre-APC(min/+) mice, in which Atg7 had been conditionally deleted in the intestinal epithelium, were generated by mating Atg7flox/flox:vil-cre mice with APC(min/+) mice. However, there was no significant difference in the number of intestinal polyps between the Atg7flox/flox:vil-cre-APC(min/+) and the corresponding control Atg7flox/flox-APC(min/+) mice. These results indicate that autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors, and future research should focus on regulating autophagy as a form of cancer therapy.     

Versican Facilitates Chondrocyte Differentiation and Regulates Joint Morphogenesis

Versican/PG-M is a large chondroitin sulfate proteoglycan in the extracellular matrix, which is transiently expressed in mesenchymal condensation areas during tissue morphogenesis. Here, we generated versican conditional knock-out mice Prx1-Cre/Vcan^flox/flox, in which Vcan is pruned out by site-specific Cre recombinase driven by the Prx1 promoter. Although Prx1-Cre/Vcan^flox/flox mice are viable and fertile, they develop distorted digits. Histological analysis of newborn mice reveals hypertrophic chondrocytic nodules in cartilage, tilting of the joint, and a slight delay of chondrocyte differentiation in digits. By immunostaining, whereas the joint interzone of Prx1-Cre/Vcan^+/+ shows an accumulation of TGF-β, concomitant with versican, that of Prx1-Cre/Vcan^flox/flox without versican expression exhibits a decreased incorporation of TGF-β. In a micromass culture system of mesenchymal cells from limb bud, whereas TGF-β and versican are co-localized in the perinodular regions of developing cartilage in Prx1-Cre/Vcan^+/+, TGF-β is widely distributed in Prx1-Cre/Vcan^flox/flox. These results suggest that versican facilitates chondrogenesis and joint morphogenesis, by localizing TGF-β in the extracellular matrix and regulating its signaling.     

Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5(-/-) mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5( flox/flox);Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

Acquired Deficiency of A20 Results in Rapid Apoptosis,Systemic Inflammation,and Abnormal Hematopoietic Stem Cell Function

A20 is a negative regulator of NF-κB, and mutational loss of A20 expression is involved in the pathogenesis of autoimmune diseases and B-cell lymphomas. To clarify the role of A20 in adult hematopoiesis, we generated conditional A20 knockout mice (A20^flox/flox) and crossed them with Mx–1Cre (MxCre ⁺) and ERT2Cre (ERT2Cre ⁺) transgenic mice in which Cre is inducibly activated by endogenous interferon and exogenous tamoxifen, respectively. A20^flox/flox MxCre ⁺ (A20Mx) mice spontaneously exhibited myeloid proliferation, B cell apoptosis, and anemia with overproduction of pro-inflammatory cytokines. Bone marrow transplantation demonstrated that these changes were caused by hematopoietic cells. NF-κB was constitutively activated in A20Mx hematopoietic stem cells (HSCs), which caused enhanced cell cycle entry and impaired repopulating ability. Tamoxifen stimulation of A20^flox/flox ERT2Cre ⁺ (A20ERT2) mice induced fulminant apoptosis and subsequent myeloproliferation, lymphocytopenia, and progressive anemia with excessive production of pro-inflammatory cytokines, as observed in A20Mx mice. These results demonstrate that A20 plays essential roles in the homeostasis of adult hematopoiesis by preventing apoptosis and inflammation. Our findings provide insights into the mechanism underlying A20 dysfunction and human diseases in which A20 expression is impaired.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon <Emphasis Type="Italic">Tyr-Cre</Emphasis>-Mediated Deletion of the Autophagy Gene <Emphasis Type="Italic">Atg7</Emphasis>

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 Atg7^f/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 Atg7^f/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 Atg7^f/f Tyr-Cre mice relative to Atg7^f/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. Atg7^f/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 Atg7^f/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.