Targeting Protein Quality Control Pathways in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a progressive neurodegenerative disease caused by a CAG/glutamine (polyQ) expansion in the androgen receptor (AR). Like many other age-dependent neurodegenerative diseases, SBMA is characterized by the buildup of misfolded proteins into nuclear aggregates and neurodegeneration. The mutant protein disrupts several cellular pathways, and decreasing levels of disease causing protein may circumvent several of the downstream pathological processes. Here we investigate the effects of manipulating protein quality control pathways in cell and animal models of SBMA, identifying novel therapeutic targets and advancing our understanding of molecular chaperones and their role in protein triage. Cells degrade proteins through two main pathways, autophagy and the ubiquitin proteasome pathway. Autophagy degrades cytosolic proteins in bulk, and increased autophagy has been shown to be beneficial in some models of protein aggregation diseases. Our results however, show that activating autophagy increases muscle wasting, while inhibiting autophagy significantly increases the lifespan and size of muscle fibers in a mouse model of SBMA. Our findings are surprising, and suggest that activation of autophagy in SBMA may exacerbate disease progression. The Hsp90/Hsp70-based chaperone machinery regulates the stabilization and degradation of Hsp90 clients through the proteasome, and presents an alternative therapeutic target to modulate proteostasis. Little is known however, about how this machinery functions to triage misfolded proteins, and few modulators of Hsp70 exist. Here we advance our understanding of chaperone machinery function, and present novel strategies to target Hsp70’s substrate affinity. We demonstrate that inhibiting Hsp70 function leads to accumulation of toxic AR, while increasing Hsp70 substrate affinity through overexpression of the co-chaperone Hip, or through treatment with a newly identified small molecule allosteric activator, promotes client protein ubiquitination and polyQ AR clearance. Both genetic and pharmacologic approaches to increase Hsp70 activity rescue disease phenotype in a Drosophila model of SBMA. Our results reveal a new therapeutic strategy of targeting Hsp70 to treat SBMA and perhaps other neurodegenerative diseases, while providing insights into the role of the chaperone machinery in protein quality control.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91413/1/amwang_1.pd

The X-Ray Position and Infrared Counterpart of the Eclipsing X-Ray Pulsar OAO 1657-415

We have measured the precise position of the 38-s eclipsing X-ray pulsar OAO 1657-415 with the Chandra X-Ray Observatory: RA = 17h00m48.90s, Dec = -41d39m21.6s, equninox J2000, error radius = 0.5 arcsec. Based on the previously measured pulsar mass function and X-ray eclipse duration, this 10.4-d high-mass X-ray binary is believed to contain a B supergiant companion. Deep optical imaging of the field did not detect any stars at the Chandra source position, setting a limit of V>23. However, near-IR imaging revealed a relatively bright star (J=14.1, H=11.9, K_s=10.7) coincident with the Chandra position, and we identify this star as the IR counterpart of OAO 1657-415. The IR colors and magnitudes and the optical non-detections for this star are all consistent with a highly reddened B supergiant (A_V= 20.4 +/- 1.3) at a distance of 6.4 +/- 1.5 kpc. This implies an X-ray luminosity of 3e36 erg/s (2-10 keV). IR spectroscopy can verify the spectral type of the companion and measure its radial velocity curve, yielding a neutron star mass measurement.Comment: 4 pages. ApJ in press (Vol. 573, July 10 issue

Isolation site influences virulence phenotype of serotype 14 Streptococcus pneumoniae strains belonging to multilocus sequence type 15

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains

A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease

Factors Predictive of Being Bullies or Victims of Bullies in US Elementary Schools.

We analyzed a population-representative cohort (N=13,611; Mage at kindergarten, first, and second grade = 67.5, 79.5, and 91.5 months, respectively) to identify kindergarten to second grade factors predictive of being bullies or victims during third to fifth grade. We did so by estimating a block recursive structural equation model (SEM) with three sets of predictors. These were: (a) individual and school socio-demographics; (b) family distress and harsh parenting; and (c) individual behavior and achievement. Relations between each of the included variables and the bullying outcomes were simultaneously estimated within the SEM. Thus, each variable served as a control for estimating the effects of the other variables. We used robust standard errors to account for student clustering within schools. Results indicated that externalizing problem behavior strongly predicted being a bully ([ES] = .56, p\u3c.001) and a victim (ES=.29, p\u3c.001). We observed a negative relation between being Hispanic and being a victim (ES = −.10, p\u3c.001) and a positive relation between being Black and being a bully (ES = .11, p\u3c.001). We also observed statistically significant relations between a family’s socioeconomic status and being a bully (ES = −.08, p\u3c.001) as well as school poverty and being a victim (ES = .07, p\u3c.001). The results advance the field’s limited understanding of risk and protective factors for bullying perpetration or victimization during elementary school and provide additional empirical support for assisting young children already exhibiting externalizing problem behaviors

Macroautophagy Is Regulated by the UPR–Mediator CHOP and Accentuates the Phenotype of SBMA Mice

Altered protein homeostasis underlies degenerative diseases triggered by misfolded proteins, including spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by a CAG/glutamine expansion in the androgen receptor. Here we show that the unfolded protein response (UPR), an ER protein quality control pathway, is induced in skeletal muscle from SBMA patients, AR113Q knock-in male mice, and surgically denervated wild-type mice. To probe the consequence of UPR induction, we deleted CHOP (C/EBP homologous protein), a transcription factor induced following ER stress. CHOP deficiency accentuated atrophy in both AR113Q and surgically denervated muscle through activation of macroautophagy, a lysosomal protein quality control pathway. Conversely, impaired autophagy due to Beclin-1 haploinsufficiency decreased muscle wasting and extended lifespan of AR113Q males, producing a significant and unexpected amelioration of the disease phenotype. Our findings highlight critical cross-talk between the UPR and macroautophagy, and they indicate that autophagy activation accentuates aspects of the SBMA phenotype

Which Children are Frequently Victimized in U.S. Elementary Schools? Population-based Estimates

We analyzed a population-based cohort of 11,780 U.S. kindergarten children to identify risk and protective factors predictive of frequent verbal, social, reputational, and/or physical bullying victimization during the upper elementary grades. We also stratified the analyses by biological sex. Both girls and boys displaying kindergarten externalizing problem behaviors were at consistently higher risk of frequent victimization during 3rd-5th grade (for the combined sample of boys and girls, verbal odds ratio [OR] = 1.82, social OR = 1.60, reputational OR = 1.85, physical OR = 1.67, total OR = 1.93). Hispanic children relative to non-Hispanic White children and those from higher income families were the most strongly and consistently protected from victimization. Boys were more likely to be physically bullied but less likely to be verbally, socially or reputationally bullied than girls. Other variables including disability, cognitively stimulating parenting, academic achievement, and internalizing behavior problems had statistically significant but less consistent and generally weaker relations with frequent victimization

T Cell Detection of a B-Cell Tropic Virus Infection: Newly-Synthesised versus Mature Viral Proteins as Antigen Sources for CD4 and CD8 Epitope Display

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36–48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells

Genome-Wide Association Study Identifies Loci for Liver Enzyme Concentrations in Mexican Americans: The GUARDIAN Consortium.

ObjectivePopulations of Mexican American ancestry are at an increased risk for nonalcoholic fatty liver disease. The objective of this study was to determine whether loci in known and novel genes were associated with variation in aspartate aminotransferase (AST) (n = 3,644), alanine aminotransferase (ALT) (n = 3,595), and gamma-glutamyl transferase (GGT) (n = 1,577) levels by conducting the first genome-wide association study (GWAS) of liver enzymes, which commonly measure liver function, in individuals of Mexican American ancestry.MethodsLevels of AST, ALT, and GGT were determined by enzymatic colorimetric assays. A multi-cohort GWAS of individuals of Mexican American ancestry was performed. Single-nucleotide polymorphisms (SNP) were tested for association with liver outcomes by multivariable linear regression using an additive genetic model. Association analyses were conducted separately in each cohort, followed by a nonparametric meta-analysis.ResultsIn the PNPLA3 gene, rs4823173 (P = 3.44 × 10-10 ), rs2896019 (P = 7.29 × 10-9 ), and rs2281135 (P = 8.73 × 10-9 ) were significantly associated with AST levels. Although not genome-wide significant, these same SNPs were the top hits for ALT (P = 7.12 × 10-8 , P = 1.98 × 10-7 , and P = 1.81 × 10-7 , respectively). The strong correlation (r2  = 1.0) for these SNPs indicated a single hit in the PNPLA3 gene. No genome-wide significant associations were found for GGT.ConclusionsPNPLA3, a locus previously identified with ALT, AST, and nonalcoholic fatty liver disease in European and Japanese GWAS, is also associated with liver enzymes in populations of Mexican American ancestry