Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis

The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential–mucolipin-1 (TRP-ML1) ion channel. The “biogenesis” model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV

Predictive factors for overactive bladder symptoms after pelvic organ prolapse surgery

Contains fulltext : 89696.pdf (publisher's version ) (Closed access)INTRODUCTION AND HYPOTHESIS: This study focussed on the factors which predict the presence of symptoms of overactive bladder (OAB) after surgery for pelvic organ prolapse (POP). METHODS: Consecutive women who underwent POP surgery with or without the use of vaginal mesh materials in the years 2004-2007 were included. Assessments were made preoperatively and at follow-up, including physical examination (POP-Q) and standardised questionnaires (IIQ, UDI and DDI). RESULTS: Five hundred and five patients were included with a median follow-up of 12.7 (6-35) months. Bothersome OAB symptoms decreased after POP surgery. De novo bothersome OAB symptoms appeared in 5-6% of the women. Frequency and urgency were more likely to improve as compared with urge incontinence and nocturia. The best predictor for the absence of postoperative symptoms was the absence of preoperative bothersome OAB symptoms. CONCLUSION: The absence of bothersome OAB symptoms preoperatively was the best predictor for the absence of postoperative symptoms.1 september 201

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors

Midline fascial plication under continuous digital transrectal control: which factors determine anatomic outcome?

Contains fulltext : 88897.pdf (publisher's version ) (Closed access)INTRODUCTION AND HYPOTHESIS: The aim of the study was to report anatomic and functional outcome of midline fascial plication under continuous digital transrectal control and to identify predictors of anatomic failure. METHODS: Prospective observational cohort. Anatomic success defined as POP-Q stage or= II underwent midline fascial plication under continuous digital transrectal control. Median follow-up was 14 months (12-35 months), and anatomic success was 80.3% (95% CI 75-86). Independent predictors of failure were posterior compartment POP stage >or= III [OR 8.7 (95% CI 2.7-28.1)] and prior colposuspension [OR 5.6 (95% CI 1.1-27.8)]. Sixty-three percent of patients bothered by obstructed defaecation experienced relief after surgery. CONCLUSIONS: Anatomic and functional outcomes were good. Risk factors for anatomic failure were initial size of posterior POP (stage >or= III) and prior colposuspension.1 juni 201

Pelvic organ prolapse and collagen-associated disorders

Contains fulltext : 109010.pdf (publisher's version ) (Open Access)INTRODUCTION AND HYPOTHESIS: Pelvic organ prolapse (POP) and other disorders, such as varicose veins and joint hypermobility, have been associated with changes in collagen strength and metabolism. We hypothesized that these various disorders were more prevalent in both POP patients and their family members. METHODS: In this study, the prevalence of various collagen-associated disorders, including POP, was compared between POP patients (n = 110) and control patients (n = 100) and their first and second degree family members. RESULTS: POP patients reported a higher prevalence of varicose veins, joint hypermobility and rectal prolapse and were more likely to have family members with POP as compared to the control group (p < 0.01). In contrast, the family members of the POP group did not report a higher prevalence of collagen-associated disorders compared to the family members of the control group (p = 0.82). CONCLUSIONS: POP and other collagen-associated disorders may have a common aetiology, originating at the molecular level of the collagens.1 maart 201

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy

Inaugural Charles River World Congress on Animal Models in Drug Discovery and Development

https://deepblue.lib.umich.edu/bitstream/2027.42/138112/1/12967_2017_Article_1274.pd

THE ROLE OF MUCOLIPIN-1 IN PATHOGENESIS OF THE LYSOSOMAL STORAGE DISEASE MUCOLIPIDOSIS TYPE IV

Lysosomal storage diseases (LSDs) are a group of inherited disorders that are caused by the defective activity of lysosomal proteins, resulting in the intracellular accumulation of undigested lysosomal metabolites. Mucolipidosis type IV (MLIV) is a neurodegenerative, recessive disease that results in the accumulation of undigested material in most tissue types. MLIV is caused by mutations in the MCOLN1 gene, encoding the transient receptor potential (TRP) cation channel family member mucolipin-1 (TRP-ML1). While previous work has provided insight into the role of this protein in progression of MLIV, a detailed knowledge of TRP-ML1 function and subsequent role in MLIV pathogenesis remains unclear. The aim of this project was to gain a better understanding of TRP-ML1 function and to provide further insight into the molecular mechanisms behind MLIV pathogenesis. Since TRP-ML1 is lysosomally localized and is subject to proteolysis, I wanted to first examine the trafficking and characterize the processing of this protein. TRP-ML1 undergoes cleavage within its first extracellular loop during its biosynthetic delivery to lysosomes. The lysosomal delivery of TRP-ML1 is impaired by depletion of the Adaptor Protein (AP) complex AP-1, while proteolysis remains unaffected in cells lacking AP-1, indicating that cleavage may occur in a pre-lysosomal compartment. Current models of MLIV pathogenesis suggest that TRP-ML1 directly regulates the postendocytic membrane trafficking by mediating interactions between late endosomes and lysosomes. Acute down-regulation of TRP-ML1 reveals that the lysosomal delivery and degradation of various markers is unperturbed, arguing against this model for MLIV pathogenesis. However, LDL-derived cholesterol ester hydrolysis in MLIV fibroblasts was found to be selectively impaired, suggesting that loss of TRP-ML1 alters lysosomal hydrolytic activity and has a cumulative effect on lysosome function.My findings support a role for TRP-ML1 in maintenance of the ionic balance that is critical for proper lysosome function. This work is significant because it shifts focus away from the concept that TRP-ML1 only functions to regulate specific membrane trafficking events along the endocytic pathway. These results contribute to a more complete understanding of the physiological role of TRP-ML1 and broaden current understanding of MLIV progression, providing the basis for potential therapeutic treatment strategies

Activation of the Caenorhabditis elegans degenerin channel by shear stress requires the MEC-10 subunit

Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC-4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain, and extracellular domain) and sites (residues 130–132 and 134–137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel's responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel's response to mechanical forces