Activation of autophagy by inflammatory signals limits IL-1ß production by targeting ubiquitinated inflammasomes for destruction.

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1ß (IL-1ß) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration.

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2-/- mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2-/- photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2-/- and Pnpla2+/- mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.

Comparative survey of mitochondrial ultrastructure in IDH1-mutant astrocytoma and IDH1-wildtype glioblastoma (GBM).

Gliomas are the most common malignant brain tumors with poor prognosis. The WHO's classification recognizes isocitrate dehydrogenase 1 (IDH1) mutant astrocytoma and IDH1-wildtype glioblastoma (GBM). The IDH1 mutation confers a survival advantage over the wildtype. There are several explanations for the metabolic advantage of the IDH1 mutation, some involve mitochondrial implications. Since an ultrastructural comparison of both tumor genotypes is still lacking, we surveyed the ultrastructural effects of the IDH1 mutation on the mitochondria of the IDH1-mutant astrocytoma (n = 15) and IDH1-wildtype glioblastoma (n = 15) tumors. Our results show that both IDH1 genotypes have degenerate and uncoupled mitochondria; this has not been reported before. The presence of ample lipid inclusions and lipid droplets in the cytoplasm of both genotypes support our conclusion of dysfunctional uncoupled mitochondria. Thus, the IDH1 mutation may have no ultrastructural consequences on the mitochondria, and the aberrant mitochondria in both genotypes may be the result of other unknown mutations. The status of the mitochondria in these genotypes portends a clinical challenge since tumor cells with uncoupled mitochondria are more primitive, aggressive, and considerably treatment resistant.

Astrocytoma and glioblastoma IDH1-wildtype cells colonize tumor vessels and deploy vascular mimicry.

Gliomas are the most prevalent type of malignant brain tumors with a very dismal prognosis. Angiogenesis in glioma has recently gotten more attention and its molecular aspects have been published; however, these were not complemented with ultrastructural evidence. Our ultrastructural examination of glioma vessels reveals several unique and critical features related to their mechanisms of progression and metastasis strategy. The detailed ultrastructural survey of 18 isocitrate dehydrogenase-wildtype (IDH1-wt) glioblastomas and 12 isocitrate dehydrogenase-mutant (IDH1-mt) High-grade gliomas indicated that tumor vessels of both types had undergone deformities such as the thickening of the vessel wall (VW) and proliferation of the basement membrane, contour distortions, abnormal and discontinuous basal lamina, tumor cells' invasion and colonization of VW, disappearance of endothelial cells (ECs), pericytes, and smooth muscle cells, as well as the formation of a continuous ring of tumor cells attached to the luminal side of VW in numerous cases. The latter feature is a clear sign of vascular mimicry (VM) that was previously suggested in gliomas but never shown by TEM. Additionally, the vascular invasion was carried out by a large number of tumor cells and was accompanied by the accumulation of tumor lipids in the vessels' lumina and VWs; these two features are distinct for gliomas and may alter the course of the clinical presentation and overall prognosis. This raises the issue of how to specifically target tumor cells involved in vascular invasion in order to optimize prognosis and overcome these mechanisms employed by the tumor cells.

Ligand-Induced Activation of GPR110 (ADGRF1) to Improve Visual Function Impaired by Optic Nerve Injury.

It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Regulation of phagolysosomal activity by miR-204 critically influences structure and function of retinal pigment epithelium/retina.

MicroRNA-204 (miR-204) is expressed in pulmonary, renal, mammary and eye tissue, and its reduction can result in multiple diseases including cancer. We first generated miR-204-/- mice to study the impact of miR-204 loss on retinal and retinal pigment epithelium (RPE) structure and function. The RPE is fundamentally important for maintaining the health and integrity of the retinal photoreceptors. miR-204-/- eyes evidenced areas of hyper-autofluorescence and defective photoreceptor digestion, along with increased microglia migration to the RPE. Migratory Iba1+ microglial cells were localized to the RPE apical surface where they participated in the phagocytosis of photoreceptor outer segments (POSs) and contributed to a persistent build-up of rhodopsin. These structural, molecular and cellular outcomes were accompanied by decreased light-evoked electrical responses from the retina and RPE. In parallel experiments, we suppressed miR-204 expression in primary cultures of human RPE using anti-miR-204. In vitro suppression of miR-204 in human RPE similarly showed abnormal POS clearance and altered expression of autophagy-related proteins and Rab22a, a regulator of endosome maturation. Together, these in vitro and in vivo experiments suggest that the normally high levels of miR-204 in RPE can mitigate disease onset by preventing generation of oxidative stress and inflammation originating from intracellular accumulation of undigested photoreactive POS lipids. More generally, these results implicate RPE miR-204-mediated regulation of autophagy and endolysosomal interaction as a critical determinant of normal RPE/retina structure and function.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction.

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration.

The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO3 (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO3-treated wild type mice that progressed with time post-injection. NaIO3-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

PhyloOncology: Understanding cancer through phylogenetic analysis.

Despite decades of research and an enormity of resultant data, cancer remains a significant public health problem. New tools and fresh perspectives are needed to obtain fundamental insights, to develop better prognostic and predictive tools, and to identify improved therapeutic interventions. With increasingly common genome-scale data, one suite of algorithms and concepts with potential to shed light on cancer biology is phylogenetics, a scientific discipline used in diverse fields. From grouping subsets of cancer samples to tracing subclonal evolution during cancer progression and metastasis, the use of phylogenetics is a powerful systems biology approach. Well-developed phylogenetic applications provide fast, robust approaches to analyze high-dimensional, heterogeneous cancer data sets. This article is part of a Special Issue entitled: Evolutionary principles - heterogeneity in cancer?, edited by Dr. Robert A. Gatenby.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors.

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Nevirapine-induced liver lipid-SER inclusions and other ultrastructural aberrations.

Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae-lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.

Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma.

We have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.

Mutated myocilin and heterozygous Sod2 deficiency act synergistically in a mouse model of open-angle glaucoma.

Glaucoma is a multifactorial optic neuropathy characterized by retinal ganglion cell (RGC) death and axonal degeneration leading to irreversible blindness. Mutations in the myocilin (MYOC) gene are the most common genetic factors of primary open-angle glaucoma. To develop a genetic mouse model induced by the synergistic interaction of mutated myocilin and another significant risk factor, oxidative stress, we produced double-mutant mice (Tg-MYOC(Y437H/+)/Sod2(+/-)) bearing human MYOC with a Y437H point mutation and a heterozygous deletion of the gene for the primary antioxidant enzyme, superoxide dismutase 2 (SOD2). Sod2 is broadly expressed in most tissues including the trabecular meshwork (TM) and heterozygous Sod2 knockout mice exhibit the reduced SOD2 activity and oxidative stress in all studied tissues. Accumulation of Y437H myocilin in the TM induced endoplasmic reticulum stress and led to a 45% loss of smooth muscle alpha-actin positive cells in the eye drainage structure of 10- to 12-month-old Tg-MYOC(Y437H/+)/Sod2(+/-) mice as compared with wild-type littermates. Tg-MYOC(Y437H/+)/Sod2(+/-) mice had higher intraocular pressure, lost about 37% of RGCs in the peripheral retina, and exhibited axonal degeneration in the retina and optic nerve as compared with their wild-type littermates. Single-mutant littermates containing MYOC(Y437H/+) or Sod2(+/-) exhibited no significant pathological changes until 12 months of age. Additionally, we observed elevated expression of endothelial leukocyte adhesion molecule-1, a human glaucoma marker, in the TM of Tg-MYOC(Y437H/+)/Sod2(+/-) mice. This is the first reported animal glaucoma model that combines expression of a glaucoma-causing mutant gene and an additional mutation mimicking a deleterious environment factor that acts synergistically.

Loss of endothelial planar cell polarity and cellular clearance mechanisms in age-related macular degeneration.

Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch's membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.

Adverse effects of antiretroviral therapy on liver hepatocytes and endothelium in HIV patients: An ultrastructural perspective.

Human immunodeficiency virus and antiretroviral therapy (ART) together can be far more detrimental to liver cells than either of the two unaided. However, ultrastructural aspects of the synergistic effects of HIV and ART have been understudied. In a patient cohort receiving ART, this study characterizes ultrastructurally sinusoidal degeneration, hepatocytic aberrations, mitochondrial dysfunction, accumulation of bulky lipid droplets (steatosis), and occlusion of sinusoidal lumina. Mitochondrial dysfunction causes the accumulation of acetyl-CoA which leads to insulin upregulation and resistance, lipid synthesis, and steatosis. Lipid droplets deposited in the sinusoids could be the source of the blood's lipid profile alterations in HIV patients on ART.

SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome.

Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia.

Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut(-/-) mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut(-/-);Tg(INS-Alb-Mut) mice, although completely rescued from neonatal lethality that was displayed by Mut(-/-) mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut(-/-);Tg(INS-Alb-Mut) kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut(-/-);Tg(INS-Alb-Mut) mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.