Untersuchungen zum Proteom humaner lysosomaler Membranproteine sowie zum Niemann-Pick Typ C-1 Protein

Von den Proteinen der lysosomalen Membran sind bisher nur wenige bekannt. Dies steht in einem gewissen Gegensatz zu den vielfältigen spezifischen Transportaufgaben der lysosomalen Membran. Ein genetischer Defekt eines für die Aufrechterhaltung des lysosomalen Systems verantwortlichen Proteins führt zur Ausbildung einer lysosomalen Speicherkrankheit. Ein Beispiel hierfür ist die Niemann-Pick Typ C-1 Krankheit. Bei allen bisher identifizierten lysosomalen Membranproteinen handelt es sich um Glykoproteine. Bisher wurden gezielt nur einzelne Proteine untersucht, nie aber das Proteom der lysosomalen Membranproteine zweidimensional aufgetrennt und charakterisiert. In der vorliegenden Arbeit konnte gezeigt werden, dass zur Trennung eines Gemisches lysosomaler Membranproteine die isoelektrische Fokussierung in Röhrchengelen mit Trägerampholyten eine deutlich bessere Ausbeute und Auftrennung lysosomaler Membranproteine erzielt werden kann, als bei der Verwendung immobilisierter pH-Gradienten. Besonders deutlich ist dies bei Proteinen mit einem Molekulargewicht höher als 30 kDa und einem isoelektrischen Punkt größer als pH 6.0. Mit Hilfe der vorliegenden, etablierten Methode ist es möglich, bis zu 500 µg lysosomalen Membranproteins zu lysieren und in einem Bereich von pH 4.0 bis 7.0 zweidimensional aufzutrennen. Die reifen lysosomalen Membranproteine weisen ein Molekulargewicht von etwa 10 kDa bis über 200 kDa auf. Eine Natriumcarbonat-Behandlung der lysosomalen Membranproteine zeigte, dass über 90 % der Proteine im Niederschlag zu finden sind. Einige Membranproteine konnten sowohl im Niederschlag als auch im Überstand detektiert werden. Es handelt sich hierbei um periphere Membranproteine. Es konnte gezeigt werden, dass weniger als ein Viertel der immunoaffinitätsgereinigten lysosomalen Membranproteine glykosyliert sind. Hiervon tragen über 80 % Neuraminsäuren und schätzungsweise 40 % N-glykosidisch verknüpfte Oligosaccharidseitenketten. Über 35 % der detektierten glykosylierten lysosomalen Membranproteine tragen sowohl N-Glykane als auch Neuraminsäuren. Das im Western-Blot nachgewiesene lysosomal assoziierte Membranprotein-2 (LAMP-2) ist im Anschluss an die Natriumcarbonat-Behandlung im Niederschlag zu detektieren, trägt sowohl N-Glykane als auch Neuraminsäuren, weist ein apparentes Molekulargewicht von 100 bis 110 kDa auf und hat einen isoelektrischen Punkt von pH 4.2 bis 4.7. In der aus humaner Plazenta isolierten lysosomalen Membran konnte das so genannte Niemann-Pick Typ C-1 Protein (NPC-1) mittels Western-Blotting nachgewiesen werden. Das reife Protein kommt in zwei Formen vor und hat ein Molekulargewicht von 173 beziehungsweise 204 kDa. Die Behandlung mit Peptid N-Glycosidase F führt zu einer Reduktion des Molekulargewichts beider Formen auf 151 kDa. Untersuchungen zum Vorkommen des NPC-1 im Verlauf des Percoll®-Gradienten zeigten, dass dieses Membranprotein nicht nur in den schweren Fraktionen des lysosomalen Kompartiments, sondern auch in leichteren Fraktionen des Gradienten, dem so genannten endosomal/lysosomalen Kompartiment detektiert werden konnte. Die schweren lysosomalen Membranen weisen im Vergleich zu den leichten lysosomalen Membranen einen relativ höheren Cholesterol- und Sphingomyelingehalt auf. Die Phospholipide bilden die stärkste Lipidgruppe der Lysosomen. Hiervon ist der häufigste Vertreter das Phosphatidylcholin. Bezogen auf das jeweilige Ausgangsmaterial liegt das Verhältnis des Cholesterols zu den Phospholipiden der leichten Fraktionen bei 0.26 und der schweren Fraktionen bei 0.44. Die zweidimensionale Auftrennung immunoaffinitätsgereinigter Membranen der schweren und leichten Fraktionen des Percoll®-Gradienten zeigt eine sehr große Übereinstimmung im Muster des jeweiligen Proteoms

Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease‐associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full‐length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α‐secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α‐secretase‐mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho‐SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid β‐peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all oluble TREM2 was 4D9‐bound. Moreover, in a mouse model for Alzheimer's disease‐related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease‐associated state

Correction of dysregulated lipid metabolism normalizes gene expression in oligodendrocytes and prolongs lifespan in female poly-GA C9orf72 mice

Clinical and genetic research links altered cholesterol metabolism with ALS development and progression, yet pinpointing specific pathomechanisms remain challenging. We investigated how cholesterol dysmetabolism interacts with protein aggregation, demyelination, and neuronal loss in ALS. Bulk RNAseq transcriptomics showed decreased cholesterol biosynthesis and increased cholesterol export in ALS mouse models (GA-Nes, GA-Camk2a GA-CFP, rNLS8) and patient samples (spinal cord), suggesting an adaptive response to cholesterol overload. Consequently, we assessed the efficacy of the cholesterol-binding drug 2-hydroxypropyl-β-cyclodextrin (CD) in a fast-progressing C9orf72 ALS mouse model with extensive poly-GA expression and myelination deficits. CD treatment normalized cholesteryl ester levels, lowered neurofilament light chain levels, and prolonged lifespan in female but not male GA-Nes mice, without impacting poly-GA aggregates. Single nucleus transcriptomics indicated that CD primarily affected oligodendrocytes, significantly restored myelin gene expression, increased density of myelinated axons, inhibited the disease-associated oligodendrocyte response, and downregulated the lipid-associated genes Plin4 and ApoD. These results suggest that reducing excess free cholesterol in the CNS could be a viable ALS treatment strategy

How effective is tetracaine 4% gel, before a venipuncture, in reducing procedural pain in infants: a randomized double-blind placebo controlled trial

BACKGROUND: Procedural pain relief is sub-optimal in neonates. Topical tetracaine provides pain relief in children. Evidence of its efficacy and safety in neonates is limited. The objective of this study was to assess the efficacy and safety of topical tetracaine on the pain response of neonates during a venipuncture. METHODS: Medically stable infants greater than or equal to 24 weeks gestation, requiring a venipuncture, were included. Following randomization and double blinding, 1.1 g of tetracaine or placebo was applied to the skin for 30 minutes. Participants received oral sucrose if they met local eligibility criteria. The venipuncture was performed according to a standard protocol. A medium effect size in the pain score (corresponding to about 2 point difference in the PIPP score) was considered clinically significant, leading to a sample size of 142 infants, with 80% statistical power. Local skin reactions and immediate adverse cardiorespiratory events were noted. The primary outcome, PIPP score at 1 minute, was analysed using an independent Student's t-test. RESULTS: One hundred and forty two infants were included, 33 +/- 4 weeks gestation, 2100 +/- 900 grams and 6 +/- 3 days of age. There was almost no difference in PIPP scores at 1 minute between groups (mean difference -0.09; 95% confidence interval [CI]: -1.68 to 1.50; P = . 91). Similarly, there were no differences in PIPP scores during the 2(nd), 3(rd )and 4th minute. Duration of cry did not differ between the groups (median difference, 0; 95% CI, -3 to 0; P = . 84). The majority of infants in both groups received sucrose 24%. Sucrose had a significant effect on the PIPP score, as assessed by an ANOVA model (p = 0.0026). Local skin erythema was observed transiently in 11 infants (7 in the tetracaine and 4 in the placebo group). No serious side effect was observed. CONCLUSION: Tetracaine did not significantly decrease procedural pain in infants undergoing a venipuncture, when used in combination with routine sucrose administration

How effective is tetracaine 4% gel, before a peripherally inserted central catheter, in reducing procedural pain in infants: a randomized double-blind placebo controlled trial [ISRCTN75884221]

BACKGROUND: Procedural pain relief is sub-optimal in infants, especially small and vulnerable ones. Tetracaine gel 4% (Ametop(®), Smith-Nephew) provides pain relief in children and larger infants, but its efficacy in smaller infants and for peripherally inserted central catheters (PICC) remains uncertain. The objective of this trial was to assess the safety and efficacy of tetracaine gel on the pain response of very low birth weight (VLBW) infants during insertion of a PICC. METHODS: Medically stable infants greater than or equal to 24 weeks gestation, requiring a non-urgent PICC, were included. Following randomization and double blinding, 1.1 g of tetracaine or placebo was applied to the skin for 30 minutes. The PICC was inserted according to a standard protocol. Pain was assessed using the Premature Infant Pain Profile (PIPP). A 3-point change in the pain score was considered clinically significant, leading to a sample size of 54 infants, with 90% statistical power. Local skin reactions and immediate adverse cardiorespiratory events were noted. The primary outcome, PIPP score at 1 minute, was analysed using an independent Student's t-test. RESULTS: Fifty-four infants were included, 27 +/- 2 weeks gestation, 916 +/- 292 grams and 6.5 +/- 3.2 days of age. Baseline characteristics were similar between groups. The mean PIPP score in the first minute was 10.88 in the treatment group as compared to 11.74 in the placebo group (difference 0.86, 95% CI -1.86, 3.58). Median duration of crying in non-intubated infants was 181 seconds in the tetracaine group compared to 68 seconds in the placebo group (difference -78, 95% CI -539, 117). Local skin erythema was observed transiently in 4 infants (3 in the treatment and 1 in the placebo group). No serious harms were observed. CONCLUSION: Tetracaine 4% when applied for 30 minutes was not beneficial in decreasing procedural pain associated with a PICC in very small infants

Long-term expansion, genomic stability and in vivo safety of adult human pancreas organoids

Abstract: Background: Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus. Results: Our chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel. Conclusions: hPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes

Changes in quality of life 1 year after intensive care: a multicenter prospective cohort of ICU survivors

BACKGROUND: With survival rates of critical illness increasing, quality of life measures are becoming an important outcome of ICU treatment. Therefore, to study the impact of critical illness on quality of life, we explored quality of life before and 1 year after ICU admission in different subgroups of ICU survivors. METHODS: Data from an ongoing prospective multicenter cohort study, the MONITOR-IC, were used. Patients admitted to the ICU in one of eleven participating hospitals between July 2016 and June 2021 were included. Outcome was defined as change in quality of life, measured using the EuroQol five-dimensional (EQ-5D-5L) questionnaire, and calculated by subtracting the EQ-5D-5L score 1 day before hospital admission from the EQ-5D-5L score 1 year post-ICU. Based on the minimal clinically important difference, a change in quality of life was defined as a change in EQ-5D-5L score of ≥ 0.08. Subgroups of patients were based on admission diagnosis. RESULTS: A total of 3913 (50.6%) included patients completed both baseline and follow-up questionnaires. 1 year post-ICU, patients admitted after a cerebrovascular accident, intracerebral hemorrhage, or (neuro)trauma, on average experienced a significant decrease in quality of life. Conversely, 11 other subgroups of ICU survivors reported improvements in quality of life. The largest average increase in quality of life was seen in patients admitted due to respiratory disease (mean 0.17, SD 0.38), whereas the largest average decrease was observed in trauma patients (mean -0.13, SD 0.28). However, in each of the studied 22 subgroups there were survivors who reported a significant increase in QoL and survivors who reported a significant decrease in QoL. CONCLUSIONS: This large prospective multicenter cohort study demonstrated the diversity in long-term quality of life between, and even within, subgroups of ICU survivors. These findings emphasize the need for personalized information and post-ICU care. TRIAL REGISTRATION: The MONITOR-IC study was registered at ClinicalTrials.gov: NCT03246334 on August 2nd 2017

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1. yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS