NBR1 acts as an autophagy receptor for peroxisomes.

Selective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62, NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates to nascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes (pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that the amphipathic α-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain are necessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J and UBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.

PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS.

Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.Abbreviations: AAA ATPases: ATPases associated with diverse cellular activities; ABCD3: ATP binding cassette subfamily D member; 3ACOX1: acyl-CoA oxidase; 1ACTA1: actin alpha 1, skeletal muscle; ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; CAT: catalase; CQ: chloroquine; Dpf: days post fertilization: FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H2O2: hydrogen peroxide; HA - human influenza hemagglutinin; HBSS: Hanks' Balanced Salt Solution; HCQ; hydroxychloroquine; KANL: lysine alanine asparagine leucine; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; MYC: MYC proto-oncogene, bHLH transcription factor; MZ: maternal and zygotic; NAC: N-acetyl cysteine; NBR1 - NBR1 autophagy cargo receptor; PBD: peroxisome biogenesis disorder; PBS: phosphate-buffered saline; PEX: peroxisomal biogenesis factor; PTS1: peroxisome targeting sequence 1; RFP: red fluorescent protein; ROS: reactive oxygen speciess; iRNA: short interfering RNA; SKL: serine lysine leucine; SLC25A17/PMP34: solute carrier family 25 member 17; Ub: ubiquitin; USP30: ubiquitin specific peptidase 30.

The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.

Mitroflow pericardial bioprosthesis: structured failure at 4.5 years.

BACKGROUND: Prosthetic heart valves are commonly used in the management of significant valvular disease. Although bioprostheses are increasingly popular, structural deterioration with cusp tears and calcification persists. METHODS: We present a case of a Mitroflow pericardial bioprosthesis (Sorin Group Inc., Mitroflow Division, Vancouver, Canada) explanted after 4.5 years from a 78-year-old woman for bioprosthesis stenosis. RESULTS: The morphological reasons for bioprosthesis failure include structural tissue deterioration with thickening of the pericardial cusps, cusp calcification, and parastent post tears resulting in significant valvular dysfunction. CONCLUSION: To the best of our knowledge, this is the first detailed morphological description of the failure modes of a surgically explanted Mitroflow (A12) pericardial bioprosthesis.

Pulmonary valve-in-valve implants: how long do they prolong reintervention and what causes them to fail?

BACKGROUND: The valve-in-valve (VinV) procedure is a minimally invasive, transcatheter, off-pump, alternative to conventional valve replacement, which uses a failing bioprosthesis to anchor a second transcatheter-delivered prosthesis. This technique appears effective for prolonging freedom from reintervention and treating early device failure. However, it is unknown as to how long reintervention can be avoided. METHODS: We present the pathological findings of a VinV explanted after 47 months, as well as the failure modes of these devices. RESULTS: The VinV approach in our case ultimately failed, likely due to the proximity of the host's tissues to the prosthetic device, resulting in a combination of pannus, calcification, and a cusp tear. CONCLUSIONS: Additional long-term follow-up of pulmonary VinV implantations is needed in order to determine the life span of VinVs and what causes them to fail.

Cystic tumor of the atrioventricular node: rare antemortem diagnosis.

BACKGROUND: The cystic tumor of the atrioventricular node (TAV) is a rare, congenital cardiac tumor, typically located at the base of the atrial septum. Histologically benign, this multicystic mass is a tumor of the conduction system and is considered the smallest tumor capable of causing sudden and unexpected death. TAV has shown a predilection for women with a mean age at presentation of 38 years. The majority of cases are diagnosed incidentally at autopsy, while antemortem surgical excision is rare, with ours being the fifth and sixth reported cases in the medical literature. METHODS: We present two cases, in 33- and 29-year-old women who were admitted for complaints of dyspnea, dizziness, palpitation or numbness, along with a review of the literature. One was known to have complete congenital heart block and ventricular septal defect, where an intraoperative transesophageal echocardiogram revealed a right atrial mass. The other patient had a right atrial mass visible on magnetic resonance imaging, which led to surgical resection and permanent pacemaker insertion. RESULTS: Histopathological examination revealed a tumor composed of cysts, some lined by squamous epithelium, and others by transitional epithelium. Irregular proliferation of glandular structures with squamoid nests within a fibrous stroma, with sebaceous-type differentiation, was also observed. A chronic inflammatory component with secondary lymphoid follicles was also noted. CONCLUSION: These cases are presented, along with a review of the four previously reported cases of TAV diagnosed antemortem. Awareness regarding this lesion could improve gross and microscopic characterization of TAV and increase antemortem diagnoses.

Characterizing the inflammatory reaction in explanted Medtronic Freestyle stentless porcine aortic bioprosthesis over a 6-year period.

BACKGROUND: The Medtronic Freestyle valve is a stentless porcine valve with reportedly excellent clinical and hemodynamic results, but little has been reported about its long-term pathology. METHODS: Seventeen Freestyle valves were explanted (from 2003 to 2009) and reviewed to assess reasons for bioprosthesis failure. All valves were examined in detail, using histochemistry and immunohistochemistry to identify morphological changes, as well as cellular and humoral responses. RESULTS: One Freestyle valve, explanted for mitral valve endocarditis on the fifth postoperative day, was excluded from analysis. The average implant duration was 71.1±35.2 months. Six valves were explanted for infective endocarditis, six for aortic insufficiency, and four for aortic stenosis. Calcification was seen in 11 explants, pannus in 15, thrombus in 12, cusp tears in 9, and 10 explants showed needle tract-like injuries. A chronic inflammatory reaction involving the xenograft arterial wall was seen in 15 of 16 valves. The cells were composed of macrophages and lymphocytes, including T cells (CD8 positive) and B cells. Significant damage to the porcine aortic wall was seen in 15 cases, and cusp myocardial shelf damage in 7 cases. All cases stained positively for IgG and C4dpar. CONCLUSIONS: The porcine aortic tissue showed T cell-mediated rejection and significant aortic medial damage, consistent with dilatation of the porcine aortic root. The demonstration of IgG suggests the likelihood of humoral rejection, in addition to cellular rejection. One of the underlying possibilities is that the porcine aortic tissues are inadequately fixed, hence the retained antigenicity.

Modes of failure in explanted mitroflow pericardial valves.

BACKGROUND: Bioprosthetic heart valves are a treatment option for patients with significant valvular disease. We present the first morphologic analysis of a series of explanted Mitroflow pericardial valves (model A12) and discuss their modes of failure. METHODS: Between 2001 and 2010, 12 Mitroflow (A12) valves (Sorin Group Inc, Vancouver, Canada) were examined, grossly and microscopically, for postimplantation changes. Cusp tears and thickening, calcification (at radiography), pannus formation, inflammation, and thrombus deposition were noted. Microscopically, tissue degeneration including the presence of calcification, pannus formation, thrombus, collagen disruption, fluid insudation, inflammatory cells, and microorganisms were looked for. Causes for dysfunction were then identified. RESULTS: There were 12 patients with a mean age of 73.0 ± 5.3 years at implantation. Duration of implantation ranged from 0.25 to 7 years (mean = 2.9 ± 2.5 years). Five valves were explanted for infective endocarditis or thrombotic vegetations. Primary structural deterioration was observed in 7 of 12 (58%) valves; characterized by para-stent post cusp tears (42%) and moderate to severe calcification (50%). Most valves showed cusp thickening and collagen disruption at the stent post and pannus. CONCLUSIONS: Structural valve deterioration was observed in 100% of the Mitroflow pericardial valves that had been in place for more than 2 years. The high rate of para-stent post cusp tears, calcification, and tissue degeneration suggests a link to the design, likely associated with mechanical stress, in addition to abrasion with the cross-sutures and Dacron fabric at the stent posts.