mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Rab12 is a regulator of LRRK2 and its activation by damaged lysosomes.

Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.

Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson's disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2. Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson's disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear. To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson's disease-linked mutations (one in LRRK2 itself and one in another lysosomal regulator, VPS35), suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease. Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson's disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson's disease.

A Fixable Fluorescence-Quenched Substrate for Quantitation of Lysosomal Glucocerebrosidase Activity in Both Live and Fixed Cells.

Fluorogenic substrates are emerging tools that enable studying enzymatic processes within their native cellular environments. However, fluorogenic substrates that function within live cells are generally incompatible with cellular fixation, preventing their tandem application with fundamental cell biology methods such as immunocytochemistry. Here we report a simple approach to enable the chemical fixation of a dark-to-light substrate, LysoFix-GBA, which enables quantification of glucocerebrosidase (GCase) activity in both live and fixed cells. LysoFix-GBA enables measuring responses to both chemical and genetic perturbations to lysosomal GCase activity. Further, LysoFix-GBA permits simple multiplexed co-localization studies of GCase activity with subcellular protein markers. This tool will aid studying the role of GCase activity in Parkinson's Disease, creating new therapeutic approaches targeting the GCase pathway. This approach also lays the foundation for an approach to create fixable substrates for other lysosomal enzymes.

Functional genomics of OCTN2 variants informs protein-specific variant effect predictor for Carnitine Transporter Deficiency.

Genetic variants in SLC22A5, encoding the membrane carnitine transporter OCTN2, cause the rare metabolic disorder Carnitine Transporter Deficiency (CTD). CTD is potentially lethal but actionable if detected early, with confirmatory diagnosis involving sequencing of SLC22A5. Interpretation of missense variants of uncertain significance (VUSs) is a major challenge. In this study, we sought to characterize the largest set to date (n = 150) of OCTN2 variants identified in diverse ancestral populations, with the goals of furthering our understanding of the mechanisms leading to OCTN2 loss-of-function (LOF) and creating a protein-specific variant effect prediction model for OCTN2 function. Uptake assays with 14C-carnitine revealed that 105 variants (70%) significantly reduced transport of carnitine compared to wild-type OCTN2, and 37 variants (25%) severely reduced function to less than 20%. All ancestral populations harbored LOF variants; 62% of green fluorescent protein (GFP)-tagged variants impaired OCTN2 localization to the plasma membrane of human embryonic kidney (HEK293T) cells, and subcellular localization significantly associated with function, revealing a major LOF mechanism of interest for CTD. With these data, we trained a model to classify variants as functional (>20% function) or LOF (<20% function). Our model outperformed existing state-of-the-art methods as evaluated by multiple performance metrics, with mean area under the receiver operating characteristic curve (AUROC) of 0.895 ± 0.025. In summary, in this study we generated a rich dataset of OCTN2 variant function and localization, revealed important disease-causing mechanisms, and improved upon machine learning-based prediction of OCTN2 variant function to aid in variant interpretation in the diagnosis and treatment of CTD.

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

Microglial NF-κB drives tau spreading and toxicity in a mouse model of tauopathy.

Activation of microglia is a prominent pathological feature in tauopathies, including Alzheimer's disease. How microglia activation contributes to tau toxicity remains largely unknown. Here we show that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, activated by tau, drives microglial-mediated tau propagation and toxicity. Constitutive activation of microglial NF-κB exacerbated, while inactivation diminished, tau seeding and spreading in young PS19 mice. Inhibition of NF-κB activation enhanced the retention while reduced the release of internalized pathogenic tau fibrils from primary microglia and rescued microglial autophagy deficits. Inhibition of microglial NF-κB in aged PS19 mice rescued tau-mediated learning and memory deficits, restored overall transcriptomic changes while increasing neuronal tau inclusions. Single cell RNA-seq revealed that tau-associated disease states in microglia were diminished by NF-κB inactivation and further transformed by constitutive NF-κB activation. Our study establishes a role for microglial NF-κB signaling in mediating tau spreading and toxicity in tauopathy.

Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.

Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.

Reimagining dots and dashes: Visualizing structure and function of organelles for high-content imaging analysis.

Organelles are responsible for biochemical and cellular processes that sustain life and their dysfunction causes diseases from cancer to neurodegeneration. While researchers are continuing to appreciate new roles of organelles in disease, the rapid development of specifically targeted fluorescent probes that report on the structure and function of organelles will be critical to accelerate drug discovery. Here, we highlight four organelles that collectively exemplify the progression of phenotypic discovery, starting with mitochondria, where many functional probes have been described, then continuing with lysosomes and Golgi and concluding with nascently described membraneless organelles. We introduce emerging probe designs to explore organelle-specific morphology and dynamics and highlight recent case studies using high-content analysis to stimulate further development of probes and approaches for organellar high-throughput screening.

Vedolizumab-induced acute pancreatitis: the first reported clinical case.

Drug-induced acute pancreatitis (DIAP) is a rare, but clinically significant diagnosis. Vedolizumab, an α4ß7 integrin inhibitor, which was approved in 2015 for treatment of moderate to severe inflammatory bowel disease, is a well-tolerated medication with a favourable safety profile and minimal serious adverse events in premarketing clinical trials. We present the first reported case of acute pancreatitis directly attributable to vedolizumab.

Probing the correlation of neuronal loss, neurofibrillary tangles, and cell death markers across the Alzheimer's disease Braak stages: a quantitative study in humans.

Clarifying the mechanisms connecting neurofibrillary tangle (NFT) neurotoxicity to neuronal dysfunction in humans is likely to be pivotal for developing effective treatments for Alzheimer's disease (AD). To model the temporal progression of AD in humans, we used a collection of brains with controls and individuals from each Braak stage to quantitatively investigate the correlation between intraneuronal caspase activation or macroautophagy markers, NFT burden, and neuronal loss, in the dorsal raphe nucleus and locus coeruleus, the earliest vulnerable areas to NFT accumulation. We fit linear regressions with each count as outcomes, with Braak score and age as the predictors. In progressive Braak stages, intraneuronal active caspase-6 positivity increases both alone and overlapping with NFTs. Likewise, the proportion of NFT-bearing neurons showing autophagosomes increases. Overall, caspases may be involved in upstream cascades in AD and are associated with higher NFTs. Macroautophagy changes correlate with increasing NFT burden from early AD stages.

Outcomes of preoperative biliary drainage from a single tertiary center: Is there still a role for plastic stents?

OBJECTIVES: Preoperative biliary drainage (PBD) can relieve symptoms of cholestasis, but carries risk of procedural complications. Metal stents have wider lumens and longer patency, although plastic stents (PS) remain in use. We reviewed the outcomes after PBD in patients with cholestasis. METHODS: Patients with symptomatic cholestasis who were likely to wait for over 2 weeks before surgery and were thus treated with PBD between January 2011 and May 2015 were included. Patients were evaluated for stenting-related complications, time interval to surgery, resection rate, improvement in bilirubin level and surgical complications. RESULTS: Forty patients underwent PBD by endoscopic retrograde cholangiopancreatography (ERCP). Of these, 36 patients received the placement of PS, one received a metal stent and the remaining three required percutaneous drainage due to unsuccessful biliary cannulation. Serum bilirubin declined from 172 µmol/L (baseline) to 14 µmol/L at 30 days (P < 0.0001). Median time interval from ERCP to surgery in all patients was 5 weeks (range 2-36 weeks). Preoperative stenting-related complications occurred in seven patients after a median of 3 weeks (range 1-6 weeks). Median time to surgery was similar in patients with and without stenting-related complications (5 weeks vs 4 weeks, respectively, P = 0.33). Surgery was completed in 32 (80%) patients, with a post-Whipple complication rate of 53%. CONCLUSIONS: PBD using mostly PS was effective in reducing bilirubin levels and did not detrimentally affect time interval to surgery. Median time interval to stenting-related complication occurred after 3 weeks, suggesting PS may be most useful for short-term PBD.

Sitaxsentan-induced acute severe hepatitis treated with glucocorticoid therapy.

Endothelin receptor antagonists are commonly used in the treatment of pulmonary hypertension. Sitaxsentan, a selective endothelin A receptor blocker, induces a mild transaminitis in approximately 3% to 5% of patients, but rarely an acute severe hepatitis. A case involving a 61-year-old female with sitaxsentan-induced acute severe liver failure is presented. Depite withdrawal of therapy, her liver tests failed to improve. After six weeks of monitoring, the patient was administered high-dose corticosteroids, with a good clinical and biochemical response. While endothelin receptor antagonists are postulated to cause hepatitis by inhibition of a bile salt transporter pump, an immune-mediated or idiosyncratic mechanism should be considered.

Update of cholangioscopy and biliary strictures.

Cholangioscopy remains another modality in the investigation of biliary strictures. At cholangioscopy, the "tumour vessel" sign is considered a specific sign for malignancy. Through its ability to not only visualise mucosa, but to take targeted biopsies, it has a greater accuracy, sensitivity and specificity for malignant strictures than endoscopic retrograde cholangiopancreatography guided cytopathological acquisition. Cholangioscopy however, is time consuming and costly, requires greater technical expertise, and should be reserved for the investigation of undifferentiated strictures after standard investigations have failed.

Hemobilia.

Hemobilia is an uncommon medical problem that presents in a varied fashion and is increasingly of iatrogenic origin. The diagnosis of hemobilia needs to be considered in patients presenting with upper gastrointestinal bleeding, particularly if they are jaundiced with abdominal pain in the setting of recent or previous percutaneous liver intervention or abdominal trauma. Multislice computed tomographic angiography is increasingly being used in the investigation, but transcatheter arterial embolization remains the cornerstone of managing those patients requiring intervention. The majority of patients with hemobilia will be managed supportively or with radiologic intervention; most do not require surgical intervention.

Screening for coeliac disease using anti-tissue transglutaminase antibody assays, and prevalence of the disease in an Australian community.

OBJECTIVES: To determine (i) the prevalence of positive results of anti-tissue transglutaminase (anti-tTG) antibody assays and coeliac disease (CD) in a rural Australian community; and (ii) whether confirmatory testing of a positive assay result with an alternative anti-tTG assay improved the positive predictive value of the test in population screening for CD. DESIGN: Retrospective analysis in December 2004 of stored serum samples taken in 1994-1995 from 3011 subjects in the Busselton Health Study follow-up. Assays for IgA and IgG anti-tTG antibodies were performed, and positive or equivocal samples were retested with a different commercial anti-tTG assay. Available subjects with one or more positive assay results were interviewed, had serum collected for repeat anti-tTG assays and for HLA-DQ2 and HLA-DQ8 haplotyping and, if appropriate, gastroscopy and duodenal biopsy were performed. In unavailable subjects, HLA-DQ2 and -DQ8 haplotyping was performed on stored sera. Total serum IgA levels were assessed in subjects with initially negative assay results. MAIN OUTCOME MEASURE: Prevalence of anti-tTG positivity and biopsy-proven CD. RESULTS: In 47 of 3011 serum samples (1.56%), at least one anti-tTG assay gave positive results: 31 of the subjects who provided these sera were available for clinical review, and 21 were able to have a gastroscopy. Seventeen subjects (0.56%) were diagnosed with definite CD (14 were confirmed at gastroscopy, and three unavailable subjects had three positive results of anti-tTG assays and an HLA haplotype consistent with CD); in a further 12 unavailable subjects, CD status was considered equivocal, with one or more positive anti-tTG assay results and an HLA haplotype consistent with CD. If these subjects were regarded as having CD, the prevalence of CD would be 0.96%. The positive predictive value when all three anti-tTG assays gave positive results was 94%, but fell to 45.2% with only one positive result. CONCLUSIONS: The prevalence of anti-tTG antibodies in this population is 1.56%; the prevalence of CD is at least 0.56%. The utility of a single, positive result of an anti-tTG assay in screening for CD in the community is poor, and repeat and/or collateral assessment with different assays may decrease the need for gastroscopy and distal duodenal biopsy.

Predictive and protective factors associated with upper gastrointestinal bleeding after percutaneous coronary intervention: a case-control study.

BACKGROUND: Hemorrhagic complications of acute coronary syndromes and percutaneous coronary intervention (PCI) are associated with increased mortality. Upper gastrointestinal (UGI) bleeding after PCI is a potential target for preventative strategies. OBJECTIVE: To evaluate the risk factors for UGI bleeding in a large cohort of contemporary PCI patients and assess the outcomes of medical and endoscopic management. METHOD: A case-control study evaluating UGI bleeding in the 30 days following PCI for stable angina and acute coronary syndromes, at one institution between 1998 and 2005. Cases were identified and outcomes assessed using linkage analysis of data from institutional PCI and endoscopy databases, statewide vital statistics and hospital discharge registries, and a detailed review of medical notes for each case and three matched controls. Analysis of the case and control groups for risk and protective factors was performed using the chi2 test with Fisher's exact P value and logistic regression. RESULTS: The incidence of UGI bleeding following PCI was 1.2% (70 of 5,673 patients). The etiologies of these bleeds were diverse. Risk factors for UGI bleeding were primary PCI (OR 27.80, 95% CI 6.28-123.05, P < 0.001), cardiac arrest (OR 6.17, 95% CI 1.82-20.84, P= 0.003), inotropic requirement (OR 5.85, 95% CI 1.98-17.27, P= 0.001), thienopyridine use before PCI (OR 2.40, 95% CI 1.04-5.53, P= 0.02), and advanced age (OR 1.08, 95% CI 1.04-1.12, P < 0.001). Proton pump inhibitor use after PCI (OR 0.08, 95% CI 0.02-0.40, P= 0.002) was accompanied by a reduced risk of UGI bleeding. Endoscopy provided therapeutic intervention in 33% of patients. There were no serious complications of endoscopy. The 30-day mortality for cases was 11.9% and 0.5% for controls (P= 0.001). CONCLUSION: UGI bleeding after PCI is relatively common and associated with increased mortality. Those undergoing PCI for acute myocardial infarction or in the presence hemodynamic instability are at highest risk. Proton pump inhibition following PCI may reduce the bleeding risk, though when UGI bleeding occurs, therapeutic endoscopy is safe.