Mutant superoxide dismutase-1-caused pathogenesis in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their late mid-life, with fatal outcome usually within a few years. The progressive degeneration of neurons responsible for muscle movement (motor neurons) throughout the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually affects respiratory function. Most cases have no familial background (sporadic) whereas about 10% of cases have relatives affected by the disease. A substantial number of familial cases are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). Since the initial discovery of this relationship about 17 years ago, numerous workers have tried to identify the pathogenicity of mutant SOD1 but without any final agreement or consensus regarding mechanism. The experiments in this thesis have been aimed at finding common pathogenic mechanisms by analyzing transgenic mouse models expressing mutant SOD1s with widely different properties.     Mitochondrial pathology and dysfunction have been reported in both ALS patients and murine models. We used density gradient ultracentrifugation for comparison of mitochondrial partitioning of SOD1 in our transgenic models. It was found that models with high levels of mutant protein, overloaded mitochondria with high levels of SOD1-protein whereas models with wild type-like levels of mutant protein did not. No significant association of the truncation mutant G127X with mitochondria was found. Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis this is unlikely to be caused by physical association of mutant SOD1 with mitochondria.     Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the ventral horns of the spinal cord but also in the liver and kidney, although at lower levels. This showed that such signs of the disease can also be found outside the CNS.     This method was used further to characterize SOD1 inclusions with regard to the properties of mutant SOD1 and the presence of other proteins. The inclusions were found to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57-C146 being the major inclusion protein, constituting at least 50% of the protein content. Ten co-aggregating proteins were isolated, some of which were already known to be present in cellular inclusions. Of great interest was the presence of several proteins that normally reside in the endoplasmic reticulum (ER), which is in accordance with recent data suggesting that the unfolded protein response (UPR) has a role in ALS.     To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to the difference between transgenic and control mice. From 53 proteins finally identified, we found pathways such as mitochondrial function, oxidative stress, and protein degradation to be affected by the disease. We also identified a previously uncharacterized covalent SOD1 dimer.    In conclusion, the work described in this thesis suggests that mutant SOD1 affects the function of mitochondria, but not mainly through direct accumulation of SOD1 protein. It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues, mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be deleterious as motor neurons, unable to renew themselves, are dependent on proper protein folding and degradation

Elevated fecal peptidase D at onset of colitis in Galphai2-/- mice, a mouse model of IBD.

BACKGROUND:The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms. METHODS:Fecal samples were collected at onset of inflammation in Galphai2-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice. RESULTS:As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2-/- mice at different stages of disease. CONCLUSIONS:These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2

Orofacial Granulomatosis associated with Crohn's Disease : a Multicentre Case Series

Orofacial granulomatosis (OFG) is a rare syndrome that may be associated with Crohn's disease (CD). We aimed to characterise this relationship and the management options in the biologic era.This multicentre case series was supported by the European Crohn's and Colitis Organisation (ECCO), and performed as part of the Collaborative Network of Exceptionally Rare case reports (CONFER) project. Clinical data were recorded in a standardised collection form.This report includes 28 patients with OFG associated with CD: 14 males (mean age of 32 years, ±12.4 SD) and 14 females (40.3 years, ±21.0 SD). Non-oral upper gastrointestinal tract involvement was seen in 6 cases and perianal disease in 11. The diagnosis of OFG was made prior to CD diagnosis in 2 patients, concurrently in 8 and after CD diagnosis in 18. The distribution of OFG involved the lips in 16 cases and buccal mucosa in 18. Pain was present in 25 cases, with impaired swallowing or speaking in 6. Remission was achieved in 23 patients, notably with the use of anti-TNFs in 9 patients, vedolizumab in 1, ustekinumab in 1 and thalidomide in 2. A further 5 cases were resistant to therapies including anti-TNFs.OFG associated with CD may occur before, concurrently or after the diagnosis of CD. Perianal and UGI disease are common associations and there is a significant symptom burden in many. Remission can be obtained with a variety of immunosuppressive treatments, including several CD approved biologicals

Outcomes of bariatric surgery for patients with prevalent inflammatory bowel disease: A nationwide registry-based cohort study

Background: Obesity is becoming more prevalent in patients with inflammatory bowel disease. Although bariatric surgery is an effective treatment for obesity, questions remain regarding its safety and effec-tiveness for patients with inflammatory bowel disease. The aim of this study was to evaluate the safety and effectiveness of bariatric surgery in patients with inflammatory bowel disease. Method: This registry-based, propensity-matched cohort study included all patients who had primary Roux-en-Y gastric bypass or sleeve gastrectomy in Sweden from January 2007 to June 2020 who had an inflammatory bowel disease diagnosis and matched control patients without an inflammatory bowel disease diagnosis. The study included data from the Scandinavian Obesity Surgery Registry, the National Patient Register, the Swedish Prescribed Drugs Register, the Total Population Register, and the Education Register from Statistics Sweden.Results: In total, 71,093 patients who underwent bariatric surgery, including 194 with Crohns disease and 306 with ulcerative colitis, were 1:5 matched to non-inflammatory bowel disease control patients. The patients with Crohns disease had a higher readmission rate within 30 days (10.7% vs 6.1%, odds ratio = 1.84, 95% confidence interval 1.02-3.31) than the control patients, with no significant difference between the surgical methods. The patients with ulcerative colitis had a higher risk for serious post-operative complications after Roux-en-Y gastric bypass (8.0% vs 3.7%, odds ratio = 2.64, 95% confidence interval 1.15-6.05) but not after sleeve gastrectomy compared to control patients (0.8% vs 2.3%). No difference was observed in postoperative weight loss or postoperative health-related quality of life.Conclusion: Sleeve gastrectomy appears to be a safe and effective treatment for obesity in patients with inflammatory bowel disease, whereas Roux-en-Y gastric bypass was associated with a higher risk for postoperative complications in patients with ulcerative colitis.&amp; COPY; 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Funding Agencies| [OLL-939106]</p

Elevated fecal peptidase D at onset of colitis in Galphai2^-/- mice, a mouse model of IBD

<div><p>Background</p><p>The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms.</p><p>Methods</p><p>Fecal samples were collected at onset of inflammation in Galphai2^-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice.</p><p>Results</p><p>As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2^-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2^-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2^-/- mice at different stages of disease.</p><p>Conclusions</p><p>These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2.</p></div

Immunoblots of trypsin-2.

<p>Fecal protein samples from Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates were subjected to immunoblotting for detection of trypsin-2. (A) Equal amounts of fecal protein were loaded from Galphai2^-/- mice with early signs of colitis, along with samples from Galphai2^+/- mice and wild-type littermates of similar ages. (B) Fecal protein samples from Galphai2^-/- mice at different stages of colitis showed a tendency towards higher trypsin levels with advanced disease. The boxplots show densitometric quantitation of blots where trypsin-2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates (A) or the pre-symptomatic Galphai2^-/- mice (B). a.u. = arbitrary units.</p

PAR2 immunoblots of homogenates from colonic and pancreatic tissue of Galphai2^-/- mice and healthy littermates.

<p>To examine the possible effect of increased amounts of luminal proteases on PAR2 levels, all Galphai2^-/- mice samples loaded on gels were from mice of different stages of clinically observable colitis (> 5 weeks). <b>A.</b> PAR2 in Galphai2^-/- and wild-type mice colonic homogenates. The band of slightly lower molecular weight was a cross-reacting protein of the anti-mouse secondary antibody and was excluded from the densitometric quantitation. <b>B.</b> PAR2 in Galphai2^-/- and wild-type mice pancreatic homogenates. Relative to Galphai2^-/- the PAR2 signal was weak in wild-type mice. The boxplots show densitometric quantitation of blots where PAR2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates. a.u. = arbitrary units.</p

Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.

<p>Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.</p

Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.

<p>Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.</p

Two-dimensional electrophoresis of fecal protein samples of Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates.

<p>Samples were run on gels with broad coverage of isoelectric points (3‒11) and molecular weights. A few spots of high intensity, that markedly differed from their heterozygous and wild-type littermates, predominated in the gels from Galphai2^-/- mice.</p