Development of endothelial-targeted CD39 as a therapy for ischaemic stroke.

BACKGROUND: Ischaemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate (eATP) triggering microglial activation and causing a thromboinflammatory response culminating in endothelial activation and vascular disruption. This is further aggravated by ischaemia-reperfusion (I/R) injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates eATP by hydrolysing to adenosine which has anti-thrombotic and anti-inflammatory properties and reverses I/R injury. METHODS: We developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion (MCAo) and analysed at 24h. Anti-VCAM-CD39 or control agents (saline, non-targeted CD39, or anti-VCAM-inactive CD39) were given at 3h post-MCAo. RESULTS: Anti-VCAM-CD39 treatment reduced neurological deficit; MRI confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood brain barrier (BBB) integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tPA) further reduced infarct volumes and attenuated BBB permeability with no associated increase in intracranial haemorrhage. CONCLUSION: Anti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 h post ischaemia and may further synergise with thrombolytic therapy to improve stroke outcomes.

Examining the associations between microglia genetic capacity, early life exposures and white matter development at the level of the individual.

There are inter-individual differences in susceptibility to the influence of early life experiences for which the underlying neurobiological mechanisms are poorly understood. Microglia play a role in environmental surveillance and may influence individual susceptibility to environmental factors. As an index of neurodevelopment, we estimated individual slopes of mean white matter fractional anisotropy (WM-FA) across three time-points (age 4.5, 6.0, and 7.5 years) for 351 participants. Individual variation in microglia reactivity was derived from an expression-based polygenic score(ePGS) comprised of Single Nucleotide Polymorphisms (SNPs) functionally related to the expression of microglia-enriched genes.A higher ePGS denotes an increased genetic capacity for the expression of microglia-related genes, and thus may confer a greater capacity to respond to the early environment and to influence brain development. We hypothesized that this ePGS would associate with the WM-FA index of neurodevelopment and moderate the influence of early environmental factors.Our findings show sex dependency, where a significant association between WM-FA and microglia ePGS was only obtained for females.We then examined associations with perinatal factors known to decrease (optimal birth outcomes and familial conditions) or increase (systemic inflammation) the risk for later mental health problems.In females, individuals with high microglia ePGS showed a negative association between systemic inflammation and WM-FA and a positive association between more advantageous environmental conditions and WM-FA. The microglia ePGS in females thus accounted for variations in the influence of the quality of the early environment on WM-FA.Finally, WM-FA slopes mediated the association of microglia ePGS with interpersonal problems and social hostility in females. Our findings suggest the genetic capacity for microglia function as a potential factor underlying differential susceptibility to early life exposuresthrough influences on neurodevelopment.

Tranexamic acid in a mouse model of cerebral amyloid angiopathy: setting the stage for a novel stroke treatment approach.

Background: Symptomatic intracerebral hemorrhage (sICH) commonly occurs in patients with cerebral amyloid angiopathy (CAA). Amyloid also initiates plasminogen activation and might promote sICH. Objectives: As amyloid-driven plasmin formation can be blocked by tranexamic acid (TXA), we aimed to evaluate the biodistribution and long-term consequences of TXA on brain amyloid-beta (Aß) levels, inflammation, and neurologic function in APP/PS1 mice. Methods: APP/PS1 mice overexpressing the mutant human amyloid precursor protein and wild-type littermates were randomized to TXA (20 mg/mL) or placebo in the drinking water for 6 months. TXA in plasma and various organs was determined by liquid chromatography-mass spectrometry. Plasmin activity assays were performed to evaluate changes in fibrinolytic activity. Neurologic function was evaluated by Y-maze and parallel rod floor testing. Proximity ligation-based immunoassays were used to quantitate changes of 92 biomarkers of inflammation. Brain Aß levels were assessed by immunohistochemistry. Results: Long-term oral TXA administration inhibited fibrinolysis. TXA accumulated in the kidney (19.4 ± 11.2 µg/g) with 2- to 5-fold lower levels seen in the lung, spleen, and liver. TXA levels were lowest in the brain (0.28 ± 0.01 µg/g). Over 6 months, TXA had no discernible effect on motor coordination, novelty preference, or brain Aß levels. TXA reduced plasma levels of epithelial cell adhesion molecule and increased CCL20. Conclusion: Long-term TXA treatment does not alter brain Aß levels or impact neurologic behavior in mice predisposed to amyloid deposition and had minor effects on the levels of inflammatory mediators. This finding supports the safety of TXA and lays the foundation for TXA as a novel treatment to reduce sICH in patients with CAA.

The potential role of protease systems in hemophilic arthropathy.

Hemophilic arthropathy (HA) is characterized by joint damage following recurrent joint bleeds frequently observed in patients affected by the clotting disorder hemophilia. Joint bleeds or hemarthroses trigger inflammation in the synovial tissue, which promotes damage to the articular cartilage. The plasminogen activation system is integral to fibrinolysis, and the urokinase plasminogen activator, or uPA in particular, is strongly upregulated following hemarthroses. uPA is a serine protease that catalyzes the production of plasmin, a broad-spectrum protease that can degrade fibrin as well as proteins of the joint extracellular matrix and cartilage. Both uPA and plasmin are able to proteolytically generate active forms of matrix metalloproteinases (MMPs). The MMPs are a family of >20 proteases that are secreted as inactive proenzymes and are activated extracellularly. MMPs are involved in the degradation of all types of collagen and proteoglycans that constitute the extracellular matrix, which provides structural support to articular cartilage. The MMPs have an established role in joint destruction following rheumatoid arthritis (RA). They degrade cartilage and bone, indirectly promoting angiogenesis. MMPs are also implicated in the pathology of osteoarthritis (OA), characterized by degradation of the cartilage matrix that precipitates joint damage and deformity. HA shares a number of overlapping pathological characteristics with RA and OA. Here we discuss how the plasminogen activation system and MMPs might exacerbate joint damage in HA, lending insight into novel possible therapeutic targets to reduce the comorbidity of hemophilia.

Does an Integrated Palliative Care Program Reduce Emergency Department Transfers for Nursing Home Palliative Residents?

Background: Nursing homes (NHs) are faced with a myriad of challenges to provide quality palliative care to residents who are at their end of life. Objectives: To describe and examine the impact of the GeriCare Palliative Care Program, which comprises telemedicine, on-site clinical preceptorship, palliative care education program, and Advance Care Planning (ACP) advocacy in reducing emergency department (ED) transfers from NHs. Design: Retrospective cohort study. Setting/Subjects: A total of 217 telemedicine consults were conducted for 187 unique NH residents across 5 NHs in Singapore over a 27-month period from April 2018 to June 2020. Measurement: Records of all enrolled palliative care residents who were triaged by telemedicine consultations were examined. Results: Our findings revealed that 82% of our urgent telemedicine consultations have successfully averted ED transfers. Gender and completion of ACP were statistically significant between ED transfer group and non-ED transfer group. Among those who completed their ACP, 78.3% of the ED transfer group chose limited intervention as their main goals of care compared with 30% in the non-ED transfer group. Conclusions: The GeriCare Palliative Care Program is a novel program, which is developed to improve the quality of palliative care in NHs. The comprehensive GeriCare model comprises a systematic framework, an integration of clinical support, ACP advocacy, and education program. Our findings demonstrated that these interventions synergistically led to a reduction in ED transfers while optimizing the residents' quality of care. By carrying out the targeted initiatives to support NHs, the residents could age-in-place comfortably.

Src activates retrograde membrane traffic through phosphorylation of GBF1.

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP exchange factor (GEF) GBF1 and small GTPase Arf1. Here, we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898, are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation, and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and widespread mechanism.

The habenula clock influences response to a stressor.

The response of an animal to a sensory stimulus depends on the nature of the stimulus and on expectations, which are mediated by spontaneous activity. Here, we ask how circadian variation in the expectation of danger, and thus the response to a potential threat, is controlled. We focus on the habenula, a mediator of threat response that functions by regulating neuromodulator release, and use zebrafish as the experimental system. Single cell transcriptomics indicates that multiple clock genes are expressed throughout the habenula, while quantitative in situ hybridization confirms that the clock oscillates. Two-photon calcium imaging indicates a circadian change in spontaneous activity of habenula neurons. To assess the role of this clock, a truncated clocka gene was specifically expressed in the habenula. This partially inhibited the clock, as shown by changes in per3 expression as well as altered day-night variation in dopamine, serotonin and acetylcholine levels. Behaviourally, anxiety-like responses evoked by an alarm pheromone were reduced. Circadian effects of the pheromone were disrupted, such that responses in the day resembled those at night. Behaviours that are regulated by the pineal clock and not triggered by stressors were unaffected. We suggest that the habenula clock regulates the expectation of danger, thus providing one mechanism for circadian change in the response to a stressor.

Early Endothelial Activation in a Mouse Model of Graft vs Host Disease Following Chemotherapy.

Allogenic hematopoietic stem cell transplant (allo-HSCT) can lead to sinusoidal obstruction syndrome (SOS) and graft-versus-host disease (GvHD) in some individuals. GvHD is characterised by an immune triggered response that arises due to donor T cells recognizing the recipient tissue as "foreign". SOS results in impaired liver function due to microvascular thrombosis and consequent obstruction of liver sinusoids. Endothelial damage occurs following chemotherapy and allo-HSCT and is strongly associated with GvHD onset as well as hepatic SOS. Animal models of GvHD are rarely clinically relevant, and endothelial dysfunction remains uncharacterised. Here we established and characterised a clinically relevant model of GvHD wherein Balb/C mice were subjected to myeloablative chemotherapy followed by transplantation of bone marrow (BM) cells± splenic T-cells from C57Bl6 mice, resulting in a mismatch of major histocompatibility complexes (MHC). Onset of disease indicated by weight loss and apoptosis in the liver and intestine was discovered at day 6 post-transplant in mice receiving BM+T-cells, with established GvHD detectable by histology of the liver within 3 weeks. Together with significant increases in pro-inflammatory cytokine gene expression in the liver and intestine, histopathological signs of GvHD and a significant increase in CD4+ and CD8+ effector and memory T-cells were seen. Endothelial activation including upregulation of vascular cell adhesion molecule (VCAM)- 1 and downregulation of endothelial nitric oxide synthase (eNOS) as well as thrombosis in the liver indicated concomitant hepatic SOS. Our findings confirm that endothelial activation is an early sign of acute GvHD and SOS in a clinically relevant mouse model of GvHD based on myeloablative chemotherapy. Preventing endothelial activation may be a viable therapeutic strategy to prevent GvHD.

Characterization of a novel model of global forebrain ischaemia-reperfusion injury in mice and comparison with focal ischaemic and haemorrhagic stroke.

Stroke is caused by obstructed blood flow (ischaemia) or unrestricted bleeding in the brain (haemorrhage). Global brain ischaemia occurs after restricted cerebral blood flow e.g. during cardiac arrest. Following ischaemic injury, restoration of blood flow causes ischaemia-reperfusion (I/R) injury which worsens outcome. Secondary injury mechanisms after any stroke are similar, and encompass inflammation, endothelial dysfunction, blood-brain barrier (BBB) damage and apoptosis. We developed a new model of transient global forebrain I/R injury (dual carotid artery ligation; DCAL) and compared the manifestations of this injury with those in a conventional I/R injury model (middle-cerebral artery occlusion; MCAo) and with intracerebral haemorrhage (ICH; collagenase model). MRI revealed that DCAL produced smaller bilateral lesions predominantly localised to the striatum, whereas MCAo produced larger focal corticostriatal lesions. After global forebrain ischaemia mice had worse overall neurological scores, although quantitative locomotor assessment showed MCAo and ICH had significantly worsened mobility. BBB breakdown was highest in the DCAL model while apoptotic activity was highest after ICH. VCAM-1 upregulation was specific to ischaemic models only. Differential transcriptional upregulation of pro-inflammatory chemokines and cytokines and TLRs was seen in the three models. Our findings offer a unique insight into the similarities and differences in how biological processes are regulated after different types of stroke. They also establish a platform for analysis of therapies such as endothelial protective and anti-inflammatory agents that can be applied to all types of stroke.

ER-resident oxidoreductases are glycosylated and trafficked to the cell surface to promote matrix degradation by tumour cells.

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.

The Nuts and Bolts of Utilizing Telemedicine in Nursing Homes - The GeriCare@North Experience.

OBJECTIVES: We examined the demographics and the clinical profile of teleconsultations conducted between an acute geriatric medicine department and 8 nursing homes over a period of 6.5 years. DESIGN: This is a prospective, descriptive study. SETTING AND PARTICIPANTS: This study was conducted in 8 nursing homes of Singapore. All nursing home patients referred to the program for teleconsultation between December 2010 and May 2017 were enrolled in the study. MEASURES: The unit observed and studied was the individual telemedicine consultation. Each unique patient contributed 1 or more observation points. The data collected included consultation dates, patient demographics, background medical comorbidities, reasons for referral, physical examination findings, primary diagnoses, and general management plans recommended by the doctors. RESULTS: There were 1673 teleconsultations conducted with 850 unique patients. Most of these patients were categorized as having moderate to severe disability. Ninety-five percent of the consultations were scheduled. The main reasons for referral were for medication review (47.6%) and behavioral problems (22.1%). The 4 commonest comorbidities were hypertension (57.0%), dementia (40.0%), diabetes (39.0%), and hyperlipidemia (38%). The most common primary diagnosis was dementia (21.6%), of which 227 of those diagnosed (62.8%) were referred for behavioral and psychological symptoms of dementia. CONCLUSIONS AND IMPLICATIONS: A telemedicine service for older patients is feasible and could be of a high quality, especially if made available in places where a high density of the latter reside, such as in the care homes and other nursing and rehabilitation facilities.

Bacteria evoke alarm behaviour in zebrafish.

When injured, fish release an alarm substance (Schreckstoff) that elicits fear in members of their shoal. Although Schreckstoff has been proposed to be produced by club cells in the skin, several observations indicate that these giant cells function primarily in immunity. Previous data indicate that the alarm substance can be isolated from mucus. Here we show that mucus, as well as bacteria, are transported from the external surface into club cells, by cytoplasmic transfer or invasion of cells, including neutrophils. The presence of bacteria inside club cells raises the possibility that the alarm substance may contain a bacterial component. Indeed, lysate from a zebrafish Staphylococcus isolate is sufficient to elicit alarm behaviour, acting in concert with a substance from fish. These results suggest that Schreckstoff, which allows one individual to unwittingly change the emotional state of the surrounding population, derives from two kingdoms and is associated with processes that protect the host from bacteria.

The GalNAc-T Activation (GALA) Pathway: Drivers and markers.

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway.

Dietary Cows' Milk Protein A1 Beta-Casein Increases the Incidence of T1D in NOD Mice.

The contribution of cows' milk containing beta-casein protein A1 variant to the development of type 1 diabetes (T1D) has been controversial for decades. Despite epidemiological data demonstrating a relationship between A1 beta-casein consumption and T1D incidence, direct evidence is limited. We demonstrate that early life exposure to A1 beta-casein through the diet can modify progression to diabetes in non-obese diabetic (NOD) mice, with the effect apparent in later generations. Adult NOD mice from the F0 generation and all subsequent generations (F1 to F4) were fed either A1 or A2 beta-casein supplemented diets. Diabetes incidence in F0⁻F2 generations was similar in both cohorts of mice. However, diabetes incidence doubled in the F3 generation NOD mice fed an A1 beta-casein supplemented diet. In F4 NOD mice, subclinical insulitis and altered glucose handling was evident as early as 10 weeks of age in A1 fed mice only. A significant decrease in the proportion of non-conventional regulatory T cell subset defined as CD4⁺CD25-FoxP3⁺ was evident in the F4 generation of A1 fed mice. This feeding intervention study demonstrates that dietary A1 beta-casein may affect glucose homeostasis and T1D progression, although this effect takes generations to manifest.

Organelle Specific O-Glycosylation Drives MMP14 Activation, Tumor Growth, and Metastasis.

Cancers grow within tissues through molecular mechanisms still unclear. Invasiveness correlates with perturbed O-glycosylation, a covalent modification of cell-surface proteins. Here, we show that, in human and mouse liver cancers, initiation of O-glycosylation by the GALNT glycosyl-transferases increases and shifts from the Golgi to the endoplasmic reticulum (ER). In a mouse liver cancer model, expressing an ER-targeted GALNT1 (ER-G1) massively increased tumor expansion, with median survival reduced from 23 to 10 weeks. In vitro cell growth was unaffected, but ER-G1 strongly enabled matrix degradation and tissue invasion. Unlike its Golgi-localized counterpart, ER-G1 glycosylates the matrix metalloproteinase MMP14, a process required for tumor expansion. Together, our results indicate that GALNTs strongly promote liver tumor growth after relocating to the ER.

Digging deep into Golgi phenotypic diversity with unsupervised machine learning.

The synthesis of glycans and the sorting of proteins are critical functions of the Golgi apparatus and depend on its highly complex and compartmentalized architecture. High-content image analysis coupled to RNA interference screening offers opportunities to explore this organelle organization and the gene network underlying it. To date, image-based Golgi screens have based on a single parameter or supervised analysis with predefined Golgi structural classes. Here, we report the use of multiparametric data extracted from a single marker and a computational unsupervised analysis framework to explore Golgi phenotypic diversity more extensively. In contrast with the three visually definable phenotypes, our framework reproducibly identified 10 Golgi phenotypes. They were used to quantify and stratify phenotypic similarities among genetic perturbations. The derived phenotypic network partially overlaps previously reported protein-protein interactions as well as suggesting novel functional interactions. Our workflow suggests the existence of multiple stable Golgi organizational states and provides a proof of concept for the classification of drugs and genes using fine-grained phenotypic information.

Loss of the Habenula Intrinsic Neuromodulator Kisspeptin1 Affects Learning in Larval Zebrafish.

Learning how to actively avoid a predictable threat involves two steps: recognizing the cue that predicts upcoming punishment and learning a behavioral response that will lead to avoidance. In zebrafish, ventral habenula (vHb) neurons have been proposed to participate in both steps by encoding the expected aversiveness of a stimulus. vHb neurons increase their firing rate as expectation of punishment grows but reduce their activity as avoidance learning occurs. This leads to changes in the activity of raphe neurons, which are downstream of the vHb, during learning. How vHb activity is regulated is not known. Here, we ask whether the neuromodulator Kisspeptin1, which is expressed in the ventral habenula together with its receptor, could be involved. Kiss1 mutants were generated with CRISPR/Cas9 using guide RNAs targeted to the signal sequence. Mutants, which have a stop codon upstream of the active Kisspeptin1 peptide, have a deficiency in learning to avoid a shock that is predicted by light. Electrophysiology indicates that Kisspeptin1 has a concentration-dependent effect on vHb neurons: depolarizing at low concentrations and hyperpolarizing at high concentrations. Two-photon calcium imaging shows that mutants have reduced raphe response to shock. These data are consistent with the hypothesis that Kisspeptin1 modulates habenula neurons as the fish learns to cope with a threat. Learning a behavioral strategy to overcome a stressor may thus be accompanied by physiological change in the habenula, mediated by intrinsic neuromodulation.