Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs

Skeletal muscle mitochondrial depletion and dysfunction in chronic kidney disease

Advanced chronic kidney disease (CKD) is associated with impaired exercise capacity, skeletal muscle dysfunction, and oxidative stress. Mitochondria are the primary source for energy production and generation of reactive oxygen species (ROS). Mitochondrial state 3 respiration, mitochondrial complex I enzyme activity, and tissue porin/actin ratio were determined in the gastrocnemius muscle of male SD rats 14 weeks after 5/6 nephrectomy (CKD) or sham-operation (control). The CKD group exhibited azotemia, hypertension, significant reduction (-39%) of state 3 mitochondrial respiration, and a significant increase in the mitochondrial complex I enzyme activity. The latter is the first step in oxidative phosphorylation, a process linked to production of ROS. These abnormalities were associated with a significant reduction in muscle porin/β actin ratio denoting substantial reduction of mitochondrial mass in skeletal muscle of animals with CKD. CKD results in impaired mitochondrial respiration, reduced muscle mitochondrial mass, depressed energy production and increased ROS generation in the skeletal muscle. These events can simultaneously contribute to the reduction of exercise capacity and oxidative stress in CKD

TUNEL analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.

<p>Control 353/04 subjects’ myoblasts <b>(A)</b> untreated, <b>(B)</b> 10μM rapamycin-treated and <b>(C)</b> 10μM chloroquine-treated stained with TUNEL. VCP patients’ 421/07 myoblasts <b>(D)</b> untreated, <b>(E)</b> 10μM rapamycin-treated and <b>(F)</b> 10μM chloroquine-treated stained with TUNEL. Scale bar represents 100 μM. Data represents triplicate studies. <b>(G)</b> Percentage of TUNEL+ cells in untreated, rapamycin- and chloroquine-treated control and VCP patients’ myoblasts. Data represents triplicate studies. Statistical significance is denoted by *<i>p</i><0.005 by Student one-tailed <i>t</i>-test.</p

Immunohistochemical analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.

<p>Control 353/04 <b>(A)</b> untreated, <b>(B)</b> 10μM rapamycin-treated myoblasts, <b>(C)</b> 10μM chloroquine-treated myoblasts stained with p62/<i>SQSTM1</i> (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. VCP patient 421/07 <b>(D)</b> untreated (white arrows represent increased p62/<i>SQSTM1</i>, LC3-I/II and mislocalized TDP-43), <b>(E)</b> 10μM rapamycin-treated myoblasts (arrows indicated decreased expression levels of p62/<i>SQSTM1</i>, LC3-I/II and nuclear TDP-43), <b>(F)</b> 10μM chloroquine-treated (white arrows represent increased p62/<i>SQSTM1</i>, LC3-I/II and mislocalized TDP-43) myoblasts stained with p62/<i>SQSTM1</i> (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. Scale bar represents 100 μM. Data represents triplicate studies.</p

Immunohistochemical analyses of autophagy signaling cascade in the quadriceps of VCP^R155H/+ and WT mice treated with autophagy-modifying drugs.

<p>Quadriceps muscles from <b>(A,B)</b> control, <b>(C,D)</b> rapamycin- and <b>(E,F)</b> chloroquine-treated 20-month old WT and VCP^{<b>R155H/+</b>} mice were stained with anti-ubiquitin, p62<i>/SQSTM1</i>, LC3-I/II, and TDP-43/ubiquitin specific antibodies, respectively (shown by arrows). Cells’ nuclei were stained with DAPI (Magnification: 630X). Scale bar represents 100 μM. <b>(G)</b> Western blot expression analysis of autophagy proteins including ubiquitin, optineurin (OPTN), p62/<i>SQSTM1</i>, VCP, LC3-I/II, TDP-43, and mTOR pathway proteins: mTOR and p70S6K. Beta actin was used as a positive control. The number of mice analyzed per experiment is 6–8.</p

Mitochondrial enzyme analyses of the VCP^R155H/+ and WT mouse quadriceps treated with rapamycin or chloroquine.

<p>Quadriceps muscles from <b>(A,D)</b> vehicle control <b>(B,E)</b> rapamycin-treated or <b>(C,F)</b> chloroquine-treated animals were stained with SDH antibody to observe mitochondrial proliferation and oxidative fibers/capacity (Black arrows point to Type II dark fibers; White arrows point to lighter fibers) and <b>(G-L)</b> Oil Red O to observe lipid droplets in WT and VCP^{<b>R155H/+</b>} mice at 20 months of age (Magnification: 400X). Black arrows point to increased Oil Red O Staining; white arrows point to diminished Oil Red O staining. <b>(M)</b> Quantification of Type II oxidative fibers with autophagy-modifying drugs. The number of mice analyzed per experiment is 8–10. Statistical significance is denoted by *<i>p</i><0.005 by Student one-tailed <i>t-</i>test.</p

TUNEL analyses of quadriceps in rapamycin- and chloroquine-treated VCP^R155H/+ and WT mice.

<p>TUNEL staining of quadriceps muscles from <b>(A,B)</b> control untreated <b>(C,D)</b> rapamycin-treated and <b>(E,F)</b> chloroquine-treated WT and VCP^{<b>R155H/+</b>} animals at 20 months of age. <b>(G)</b> Quantification of TUNEL+ cells in control and treated VCP^{<b>R155H/+</b>} and WT animals. Arrows point to TUNEL+ cells indicating cell death. Statistical significance is denoted by *<i>p</i><0.005 by Student one-tailed <i>t-</i>test. The number of animals used was n = 8-10/group.</p

Hepatic proteomic analysis revealed altered metabolic pathways in insulin resistant Akt1+/−/Akt2−/− mice

ObjectiveThe aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels.MethodsTwo-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1(+/-)/AKT2(-/-) mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway Analysis was performed for the interpretation of the biological significance of the observed proteomic changes.Results11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1(+/-)/Akt2(-/-)) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways.ConclusionOur results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance

Nucleosome Organization in Human Embryonic Stem Cells

<div><p>The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a “ground state” of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this “ground state” by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.</p></div