Impaired or deficient autophagy is believed to cause or contribute to aging, as well as a number of age-related pathologies. The exact mechanism through which alterations in autophagy induce these various pathologies is not well understood. Here we describe the creation of two in vivo mouse models that allow for the characterization of the alteration in mitochondrial function and the contribution of the corresponding oxidative stress following deletion of Atg7. Using these models we demonstrate that isolated mitochondria obtained from Atg7-/- skeletal muscle exhibit a significant defect in mitochondrial respiration. We further show that cells derived from Atg7-/- mice have an altered metabolic profile characterized by decreased resting mitochondrial oxygen consumption and a compensatory increase in basal glycolytic rates. Atg7-/-cells also exhibit evidence for increased steady state levels of reactive oxygen species. The observed mitochondrial dysfunction and oxidative stress is also evident in a mouse model where Atg7 is deleted within the pancreatic β cell. In this model, the simple administration of an antioxidant can significantly ameliorate the physiological impairment in glucose-stimulated insulin secretion. Taken together, these results demonstrate the potential role of mitochondrial dysfunction and oxidative stress in autophagy related pathology.

Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here, we demonstrate that WPBs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy, or knockdown of the essential autophagy genes Atg5 or Atg7, inhibits the in vitro secretion of VWF. Furthermore, while mice with an endothelial specific deletion of Atg7 have normal vessel architecture and capillary density, these animals exhibit impaired epinephrine-stimulated VWF release, reduced levels of high molecular weight VWF multimers and a corresponding elevation of their bleeding times. Endothelial deletion of Atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis. Thus, autophagy regulates endothelial VWF secretion and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events.

We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease. (Blood. 2012;119(1): 238-250) IntroductionPoint mutations in MYH9, the gene encoding nonmuscle myosin heavy chain II-A (NMHCII-A), underlie autosomal dominant syndromes in humans (incidence, ϳ 1 in 500 000). 1-3 The human abnormalities manifest as macrothrombocytopenia, granulocyte inclusions, progressive proteinuric renal disease, cataracts, and sensorineural deafness. Most patients have a mild bleeding tendency. Patients may develop early or late onset deafness, cataracts, and progressive glomerulosclerosis, leading to kidney failure. These syndromes, now referred to as MYH9-related diseases (MYH9-RDs), were formerly called May-Hegglin, Fechtner, Sebastian, and Epstein syndromes. 1,4 Each nonmuscle myosin II (NMII) molecule is composed of a pair of heavy chains (M r ϭ 230 000) and 2 pairs of light chains (M r 20 000 and 17 000). NMII has 3 paralogs, NMII-A, NMII-Bm and NMII-C, whose heavy chains are encoded by 3 different genes MYH9, MYH10, and MYH14, respectively, located on 3 different human chromosomes (22, 17, and 19). NMIIs are ubiquitously expressed but differ with respect to localization and expression levels in cells, although they may also overlap with each other. 5 The NMII protein is markedly asymmetric with a globular-shaped motor domain at one end containing the enzymatic activity that hydrolyzes MgATP to convert chemical energy into the mechanical translocation of actin filaments. The other end of the molecule is a long ␣-helical rod that dimerizes the 2 heavy chains and participates in the formation of bipolar filaments, composed of ϳ 28 molecules, 6 which are required for most NMII functions. As a motor prote...

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