👤 Sebastian Wolfgang Schultz

Women show higher levels of Alzheimer's disease (AD) pathology than men, but the implications for cognitive decline remain unclear. Determining the extent to which tau burden differentially accelerates cognitive decline in men and women will provide critical insights into sex-specific pathways of disease progression. We leveraged tau positron emission tomography (PET), amyloid beta (Aβ) PET, apolipoprotein E (APOE) ε4 genotyping, and longitudinal cognitive data over approximately 8.6 (standard deviation [SD] = 3.8) years from 1007 cognitively unimpaired adults across three cohorts. Cognitive trajectories were modeled with linear mixed-effects regression including sex × tau × time interactions, and results were synthesized using random-effects meta-analysis. Higher tau burden in medial and lateral temporal regions was associated with faster cognitive decline in women than in men. High tau burden carries a disproportionately greater cognitive cost for women, underscoring the need for sex-specific approaches to early detection and therapeutic intervention in AD. A meta-analysis across three independent cohorts shows that female cognitive advantage at low tau shifts to vulnerability at higher tau. Sex differences in tau-related cognitive decline were consistent after accounting for amyloid burden. Sex-specific rates of cognitive decline should be considered in clinical trial design. Show less

Pancreatic alpha cells modulate beta cell function in a paracrine manner through the release of glucagon. However, the detailed molecular architecture underlying alpha-to-beta cell regulation remains poorly characterized. Here, we show that the glucagon-like peptide-1 receptor (GLP1R) is enriched as nanodomains on beta cell membranes that contact alpha cells, in keeping with increased single-molecule transcript expression. At low glucose, beta cells next to alpha cells directly sense micromolar glucagon release by pre-internalizing GLP1R. Pre-internalized GLP1R is associated with earlier beta cell Ca Show less

The surgical management of complicated diverticulitis varies across Europe. EAES members prioritized this topic to be addressed by a clinical practice guideline through an online questionnaire. To develop evidence-informed clinical practice recommendations for key stakeholders involved in the treatment of complicated diverticulitis; to improve operative and perioperative outcomes, patient experience and quality of life through a systematic evidence-to-decision approach by a diverse, multidisciplinary panel. Informed by a linked individual participant data network meta-analysis of resection and primary anastomosis (PRA) versus Hartmann's resection (HR) versus laparoscopic lavage (LPL), a panel of general and colorectal surgeons, patient partners, trialists, and fellows appraised the certainty of the evidence using GRADE and CINeMA. The panel discussed the evidence using the evidence-to-decision framework during a synchronous consensus meeting. An asynchronous modified Delphi survey was used to establish consensus. The panel suggests that patients with complicated diverticulitis without sepsis receive PRA over HR or LPL when there is availability of a surgeon with skills and experience in colorectal surgery. HR is suggested over PRA or LPL in the subgroups of septic, frail, as well as immunocompromised patients. These recommendations apply to patients with an indication for surgery. Surgeons and patients should first consider conditionally recommended interventions, then conditionally recommended against. Based on the evidence, the key benefit of PRA was a higher likelihood of not having a stoma at 1 year, with similar risks across comparisons. Conditional recommendations call for shared decision-making when considering management options. The full guideline with user-friendly decision aids is available in https://app.magicapp.org/#/guideline/7490 . This clinical practice guideline provides evidence-informed recommendations on the management of patients with complicated diverticulitis in accordance with the highest methodological standards through a structured framework informed by an international, multidisciplinary panel of stakeholders. Show less

We performed a systematic review and network meta-analysis (NMA) of individualized patient data (IPD) to inform the development of evidence-informed clinical practice recommendations. We searched MEDLINE, Embase, and Cochrane Central in October 2023 to identify RCTs comparing Hartmann's resection (HR), primary resection and anastomosis (PRA), or laparoscopic peritoneal lavage (LPL) among patients with class Ib-IV Hinchey diverticulitis. Outcomes of interest were prioritized by an international, multidisciplinary panel including two patient partners. Article screening, data extraction for IPD, and risk of bias appraisal were performed by two reviewers. We used a random-effects NMA to synthesize direct and indirect evidence. Heterogeneity was evaluated using the I Fourteen reports of seven RCTs were derived from 4,659 articles. IPD data were available for 595/678 patients (88.8%) across trials. Patients had a mean age ± SD of 64.61 ± 13.64 years and a mean BMI ± SD of 26.12 ± 5.20 kg/m PRA likely confers a lower stoma rate at 1 year compared to HR, while there may be no difference in 30-day/in-hospital mortality. LPL likely confers a higher in-hospital/30-day mortality rate compared to HR and PRA. Show less

Inflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease-specific traits during adverse remodeling, we profiled interstitial cells in early murine HFpEF using single-cell RNAseq (scRNAseq). Diastolic dysfunction and perivascular fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Integration of fibroblasts from HFpEF with two murine models for heart failure with reduced ejection fraction (HFrEF) identified a catalog of conserved fibroblast phenotypes across mouse models. Moreover, HFpEF-specific characteristics included induced metabolic, hypoxic and inflammatory transcription factors and pathways, including enhanced expression of Angiopoietin-like 4 (Angptl4) next to basement membrane compounds, such as collagen IV (Col4a1). Fibroblast activation was further dissected into transcriptional and compositional shifts and thereby highly responsive cell states for each HF model were identified. In contrast to HFrEF, where myofibroblast and matrifibrocyte activation were crucial features, we found that these cell states played a subsidiary role in early HFpEF. These disease-specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes. Furthermore, we identified a potential cross-talk between macrophages and fibroblasts via SPP1 and TNFɑ with estimated fibroblast target genes including Col4a1 and Angptl4. Treatment with recombinant ANGPTL4 ameliorated the murine HFpEF phenotype and diastolic dysfunction by reducing collagen IV deposition from fibroblasts in vivo and in vitro. In line, ANGPTL4, was elevated in plasma samples of HFpEF patients and particularly high levels associated with a preserved global-longitudinal strain. Taken together, our study provides a comprehensive characterization of molecular fibroblast activation patterns in murine HFpEF, as well as the identification of Angiopoietin-like 4 as central mechanistic regulator with protective effects. Show less

Infection-induced T cell responses must be properly tempered and terminated to prevent immuno-pathology. Using transgenic mice, we demonstrate that T cell intrinsic STAT1 signaling is required to curb inflammation during acute infection with Show less

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics. Show less

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template. Show less

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use. Show less

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), glucagon (GCG) receptor (GCGR), and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP-1R, GCGR, and GIPR were fused to the N-terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar in vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100-fold longer plasma half-lives. The GLP-1R mono agonist and GLP-1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter. Show less

In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. Show less

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal childhood-onset neurodegenerative disorder caused by mutations in ceroid lipofuscinosis neuronal-3 (CLN3), a hydrophobic transmembrane protein of unresolved function. Previous studies indicate blood-brain barrier (BBB) defects in JNCL, and our earlier report showed prominent Cln3 expression in mouse brain endothelium. Here we find that CLN3 is necessary for normal trafficking of the microdomain-associated proteins caveolin-1, syntaxin-6, and multidrug resistance protein 1 (MDR1) in brain endothelial cells. Correspondingly, CLN3-null cells have reduced caveolae, and impaired caveolae- and MDR1-related functions including endocytosis, drug efflux, and cell volume regulation. We also detected an abnormal blood-brain barrier response to osmotic stress in vivo. Evaluation of the plasma membrane with fluorescent sphingolipid probes suggests microdomain destabilization and enhanced fluidity in CLN3-null cells. In further work we found that application of the glycosphingolipid lactosylceramide to CLN3-deficient cells rescues protein transport and caveolar endocytosis. Last, we show that CLN3 localizes to the trans-Golgi network (TGN) and partitions with buoyant microdomain fractions. We propose that CLN3 facilitates TGN-to-plasma membrane transport of microdomain-associated proteins. Insult to this pathway may underlie BBB dysfunction and contribute to JNCL pathogenesis. Show less

Heart disease remains a leading cause of morbidity and mortality in the industrialized world. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disorder and the most common cause of sudden cardiac death. Foxm1 transcription factor (also known as HFH-11B, Trident, Win or MPP2) plays an important role in the pathogenesis of various cancers and is a critical mediator of post-injury repair in multiple organs. Foxm1 has been previously shown to be essential for heart development and proliferation of embryonic cardiomyocytes. However, the role of Foxm1 in postnatal heart development and in cardiac injury has not been evaluated. To delete Foxm1 in postnatal cardiomyocytes, αMHC-Cre/Foxm1(fl/fl) mice were generated. Surprisingly, αMHC-Cre/Foxm1(fl/fl) mice exhibited normal cardiomyocyte proliferation at postnatal day seven and had no defects in cardiac structure or function but developed cardiac hypertrophy and fibrosis late in life. The development of cardiomyocyte hypertrophy and cardiac fibrosis in aged Foxm1-deficient mice was associated with reduced expression of Hey2, an important regulator of cardiac homeostasis, and increased expression of genes critical for cardiac remodeling, including MMP9, αSMA, fibronectin and vimentin. We also found that following aortic constriction Foxm1 mRNA and protein were induced in cardiomyocytes. However, Foxm1 deletion did not exacerbate cardiac hypertrophy or fibrosis following chronic pressure overload. Our results demonstrate that Foxm1 regulates genes critical for age-induced cardiomyocyte hypertrophy and cardiac fibrosis. Show less

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments. Show less