A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70

Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell.     

Clinical characteristics of patients with spondyloarthritides and HLA-B27 positive antigen

The aim of this study was to present our experiences in diagnosing spondyloarthritides (SpA), and to list the most common clinical features of HLA-B27 positive patients. The study included 65 HLA-B27 positive patients with confirmed diagnosis of ankylosing spondylitis (AS) and psoriatic arthritis (PsA) who were analyzed between 2009 and 2010 in Clinic of Internal Medicine in Osijek. The diagnosis of seronegative spondyloarthritides was based on the ASAS (Assessment in AS Working Group) classification criteria for axial and then supplemented with ASAS criteria for peripheral SpA and was confirmed by radiological techniques. For diagnosing the ankylosing spondylitis (AS), there have been applied the modified New York criteria. Radiological criteria for definite sacroiliitis according to the modified New York criteria is bilateral sacroiliitis, grade 2-4 (> or = 2) or unilateral sacroiliitis, grade 3-4. For diagnosing the psoriatic arthritis (PsA), there were used CASPAR diagnostic criteria. Other features of SpA are defined within the existing classification criteria. HLA-B27 antigen was determined by direct immune-fluorescence technique using flow cytometer. The average age of patients was 50.34 years, of whom 27 female (41.53%), 38 male (58.46%). Duration of illness was 15.79 years on average. With 75.38% of patients, there had been determined the diagnosis of AS; 24.62% of patients had the diagnosis of PsA. The most common clinical characteristics that patients had were: inflammatory back pain (pain Inflammation along the lumbosacral spine), peripheral arthritis, intermittent pain in the gluteus, sacroiliitis, enthesitis, uveitis, dactilitis.     

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.     

Disease-modifying drug initiation patterns in commercially insured multiple sclerosis patients: a retrospective cohort study

Background  

The goal of this research was to compare the demographics, clinical characteristics and treatment patterns for newly diagnosed multiple sclerosis (MS) patients in a commercial managed care population who received disease-modifying drug (DMD) therapy versus those not receiving DMD therapy.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

ENDOCYTOSIS DURING POSTNATAL DIFFERENTIATION IN SUPERFICIAL CELLS OF THE MOUSE URINARY BLADDER EPITHELIUM

This electron microscopical study was performed in order to follow the endocytic pathway of horseradish peroxidase and colloidal gold tracers and to determine the involvement of endocytosis in postnatal differentiation in superficial cells of the mouse urinary bladder epithelium. Morphometric analyses of late endosomes/multivesicular bodies from day of birth to day 25 were performed. The internalisation and intracellular transport of luminal plasmalemma to multivesicular bodies via endocytic vesicles, early endosomes and pleomorphic compartments was established. Dynamic changes in endocytic activity took place within the first few days of postnatal differentiation. During this period the number of multivesicular bodies changed in an inverse ratio to their size. After the third day endocytic activity gradually approached the low rate of adult urothelium.     

Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths

Leading edge cells, which are located at the forefront of a wound margin, play a significant role in coordinating the wound healing process. In this study, leading edge cells of the urothelial explant outgrowth, resembling leading edge cells during urothelial full-thickness wound healing in vivo, were analyzed for expression and distribution of junction and differentiation-related proteins. Ultrastructural and immunofluorescence studies revealed that urothelial cells at the leading edge expressed ZO-1, claudin-4, occludin, E-cadherin, cytokeratin 7 and cytokeratin 20, while no expression of claudin-8 was noted. ZO-1, claudin-4, occludin and E-cadherin were localized along the cell membranes where neighbouring leading edge cells were in contact. Cytokeratin 7 was detected as filaments and cytokeratin 20 as small dots and sparse filaments. In conclusion, we detected early expression of ZO-1, claudin-4 and occludin at the urothelial leading edge, predicating the later formation of tight junctions as a necessary stage for the differentiation process that subsequently begins. The expression of occludin and cytokeratin 20 in urothelial cells at the leading edge suggests that leading edge cells may develop into fully differentiated superficial cells.     

Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation

Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.     

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three‐dimensional localization using confocal microscopy of immunofluorescence‐labelled GA‐related markers [GM130 (cis‐Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation‐related markers (UPs), MTs (microtubules; α‐tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non‐differentiated, UP‐negative UCs the GA is mostly organized as a single ribbon‐like structure close to the nucleus, whereas in differentiated, UP‐positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans‐Golgi/TGN enzyme β1,4‐galactosyltransferase) fused to fluorescent protein showed that Golgi‐resident enzyme cycles freely within ribbon‐like GA but not within fragmented GA. By CLEM (correlative light—electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. Conclusions. The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood—urine barrier.