A Non-Canonical Role for Choline Acetyltransferase in Chromatin Organization and the Response to Beta-Amyloid

The three-dimensional structure of chromatin is essential for context-dependent regulation of gene expression in post-mitotic neurons. Chromosomal rearrangements have been observed in the aging brain, and proteins involved in chromatin organization have altered expression and/or localization in Alzheimer’s disease (AD). A human- and primate-specific transcript of choline acetyltransferase produces an 82-kDa protein (82-kDa ChAT) that is localized to the nucleus of cholinergic neurons, but is found in the cytoplasm in individuals with mild cognitive impairment (MCI) and AD. The function of the 82-kDa ChAT protein is unknown, though recent evidence suggests it has a role in gene expression changes in response to cellular perturbations. In the present study, we explore whether 82-kDa ChAT is involved in global chromatin organization and an epigenetic response to cytotoxic amyloid-β(Aβ) exposure. We show that 82-kDa ChAT associates with chromatin in human SH-SY5Y neural cells using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), finding that acute exposure of cells to oligomeric Aβ1–42 increases 82-kDa ChAT associations with gene promoters and introns. Following Aβ1–42-exposure, 82-kDa ChAT co-localizes in nuclear aggregates with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 has similar increases in genic associations following Aβ1–42-exposure, and both SATB1 and 82-kDa ChAT associate with synapse-related genes. The 82-kDa ChAT and SATB1 proteins have patterned genomic associations at regions enriched with S/MAR binding motifs, preventing an Aβ1–42-induced increase in an isoform-specific APP mRNA transcript. Finally, we show that 82-kDa ChAT expression during cholinergic differentiation of SH-SY5Y cells increases the steady-state levels of proteins related to synapse formation, resulting in increased neurite complexity. These results demonstrate that 82-kDa ChAT and SATB1 regulate chromatin organization at S/MARs, resulting in context-dependent gene expression changes in cholinergic cells and increased expression of synapse formation-related proteins during cholinergic differentiation. Cholinergic synapse dysfunction and degeneration is observed early in AD progression and 82-kDa ChAT is mislocalized in AD, therefore the loss of both the epigenetic response to Aβ and gene expression changes related to synapse formation and maintenance may have implications for the etiology or progression of MCI and AD

The impact of teaching on the duration of common urological operations

Introduction: The ability of academic (teaching) hospitals to offer the same level of efficiency as non-teaching hospitals in a publicly funded healthcare system is unknown. Our objective was to compare the operative duration of general urology procedures between teaching and non-teaching hospitals. Methods: We used administrative data from the province of Ontario to conduct a retrospective cohort study of all adults who underwent a specified elective urology procedure (2002–2013). Primary outcome was duration of surgical procedure. Primary exposure was hospital type (academic or non-teaching). Negative binomial regression was used to adjust relative time estimates for age, comorbidity, obesity, anesthetic, and surgeon and hospital case volume. Results: 114 225 procedures were included (circumcision n=12 280; hydrocelectomy n=7221; open radical prostatectomy n=22 951; transurethral prostatectomy n=56 066; or mid-urethral sling n=15 707). These procedures were performed in an academic hospital in 14.8%, 13.3%, 28.6%, 17.1%, and 21.3% of cases, respectively. The mean operative duration across all procedures was higher in academic centres; the additional operative time ranged from 8.3 minutes (circumcision) to 29.2 minutes (radical prostatectomy). In adjusted analysis, patients treated in academic hospitals were still found to have procedures that were significantly longer (by 10‒21%). These results were similar in sensitivity analyses that accounted for the potential effect of more complex patients being referred to tertiary academic centres. Conclusions: Five common general urology operations take significantly longer to perform in academic hospitals. The reason for this may be due to the combined effect of teaching students and residents or due to inherent systematic inefficiencies within large academic hospitals

Into the Fourth Dimension: Dysregulation of Genome Architecture in Aging and Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons. Dysregulation of epigenetic processes have been observed in the aging brain and in patients with AD, though there is not yet agreement on the impact of these changes on transcription. New evidence shows that proteins involved in genome organization have altered expression and localization in the AD brain, suggesting that the genomic landscape may play a critical role in the development of AD. This review discusses the role of the chromatin organizers and epigenetic modifiers in post-mitotic cells, the aging brain, and in the development and progression of AD. How these new insights can be used to help determine disease risk and inform treatment strategies will also be discussed

Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease

Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues (14)PKLPVPP(20). We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease

The impact of common urologic complications on the risk of a periprosthetic joint infection

Copyright © 2018 By The Journal of Bone and Joint Surgery, Incorporated. Background: Periprosthetic infections after total hip arthroplasty (THA) or total knee arthroplasty (TKA) are substantial complications, and there are conflicting reports of their association with urologic complications. Our objective was to determine whether urinary tract infection (UTI) and acute urinary retention (AUR) are significant risk factors for joint infections after THA or TKA. Methods: We performed a population-based, retrospective cohort study of patients who were ≥66 years old when they underwent an initial THA or TKA between April 2003 and March 2013. Investigated exposures included a UTI presenting for treatment within 2 years after joint replacement, as well as AUR within 30 days after THA or TKA. The primary outcome was joint infection requiring hospital admission following THA or TKA (which had to occur within 2.25 years after THA or TKA for the UTI exposure or 120 days for the AUR exposure). Results: A total of 113,061 patients met the inclusion criteria and had arthroplasties (44,495 THAs and 68,566 TKAs) during the study period. The median age was 74 years (interquartile range [IQR], 70 to 79 years). Of those patients, 28,256 (25.0%) had at least 1 UTI and they were more likely to be older and female; to have had previous antibiotic exposure, cystoscopy, or urinary retention; and to have atrial fibrillation. Most of those UTIs were coded as nonspecific UTI, and the patient was seen for outpatient treatment in a non-emergency department setting. A total of 2,516 patients (2.2%) had AUR within 30 days of the procedure. Those patients were more likely to be older and male, to have medical comorbidities, to have had previous transurethral procedures or cystoscopy and previous urology visits, and to have received a general anesthetic during their procedure. A total of 1,262 patients (1.1%) had joint infection requiring hospital admission. In multivariate Cox regression analysis, UTI was associated with an increased risk of joint infection (hazard ratio [HR], 1.21 [95% confidence interval (CI), 1.14 to 1.28]; p \u3c 0.01). However multivariate analysis did not demonstrate an association between AUR and joint infection (HR, 0.99 [95% CI, 0.60 to 1.64]; p = 0.98). Conclusions: UTI was associated with increased risk of hip or knee periprosthetic joint infection, whereas AUR was not a significant risk factor. Timely and appropriate treatment of symptomatic UTIs in this patient population may be important to prevent periprosthetic joint infection

Appropriate Treatment Receipt after Breast-Conserving Surgery

Background Breast-conserving surgery (bcs) and radiation therapy (rt) are the standard of care for early breast cancer, although some women receive ipsilateral mastectomy or adjuvant tamoxifen, both of which can be appropriate alternatives to rt. Objectives of the present study were to determine the proportion of women who are treated appropriately after bcs and to identify factors associated with non-receipt of rt. Methods This retrospective cohort study used Ontario data linked at the Institute for Clinical and Evaluative Sciences to examine 33,718 patients who received bcs during 2004–2010. Primary outcome was rt receipt. The ipsilateral mastectomy rate and patient, surgeon, and setting variables were measured. Results Of the study patients, 86.1% received either rt or completion mastectomy; in the cohort less than 70 years of age, 90.8% received rt or completion mastectomy. Among patients less than 70 years of age, 3 risk factors for nonreceipt of rt were identified: age less than 46 years, treatment in a non-academic institution, and earlier year of initial bcs. Additionally, in the overall cohort, rt non-receipt was associated with high comorbidity, more than 40 km to the cancer centre, income quintile, and breast care specialization. Conclusions In Ontario, 90.8% of patients less than 70 years of age are appropriately treated for early breast cancer; approximately 1 in 10 do not receive rt or completion mastectomy. Based on those findings, women less than 46 years of age might be at increased risk of recurrence and death because of incomplete treatment. It also appears that academic centres more effectively treat breast cancer; however, breast cancer care appears to be improving over time in Ontario