Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels.

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post-COVID state. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Prevention of brain infection in the acute phase of the disease might thus be of therapeutic relevance to prevent long-lasting symptoms of COVID-19. We previously showed that melatonin or two prescribed structural analogs, agomelatine and ramelteon delay the onset of severe clinical symptoms and improve survival of SARS-CoV-2-infected K18-hACE2 mice. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevents SARS-CoV-2 entry in the brain, thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Molecular modeling analyses complemented by experimental studies in cells showed that SARS-CoV-2 entry in endothelial cells is prevented by melatonin binding to an allosteric-binding site on human angiotensin-converting enzyme 2 (ACE2), thus interfering with ACE2 function as an entry receptor for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs and its clinically used analogs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.

Simufilam Reverses Aberrant Receptor Interactions of Filamin A in Alzheimer's Disease.

Simufilam is a novel oral drug candidate in Phase 3 clinical trials for Alzheimer's disease (AD) dementia. This small molecule binds an altered form of filamin A (FLNA) that occurs in AD. This drug action disrupts FLNA's aberrant linkage to the α7 nicotinic acetylcholine receptor (α7nAChR), thereby blocking soluble amyloid beta1-42 (Aß42)'s signaling via α7nAChR that hyperphosphorylates tau. Here, we aimed to clarify simufilam's mechanism. We now show that simufilam reduced Aß42 binding to α7nAChR with a 10-picomolar IC50 using time-resolved fluorescence resonance energy transfer (TR-FRET), a robust technology to detect highly sensitive molecular interactions. We also show that FLNA links to multiple inflammatory receptors in addition to Toll-like receptor 4 (TLR4) in postmortem human AD brains and in AD transgenic mice: TLR2, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5), and T-cell co-receptor cluster of differentiation 4 (CD4). These aberrant FLNA linkages, which can be induced in a healthy control brain by Aß42 incubation, were disrupted by simufilam. Simufilam reduced inflammatory cytokine release from Aß42-stimulated human astrocytes. In the AD transgenic mice, CCR5-G protein coupling was elevated, indicating persistent activation. Oral simufilam reduced both the FLNA-CCR5 linkage and the CCR5-G protein coupling in these mice, while restoring CCR5's responsivity to C-C chemokine ligand 3 (CCL3). By disrupting aberrant FLNA-receptor interactions critical to AD pathogenic pathways, simufilam may promote brain health.

Therapeutic potential of melatonin and melatonergic drugs on K18-hACE2 mice infected with SARS-CoV-2.

As the COVID-19 pandemic grows, several therapeutic candidates are being tested or undergoing clinical trials. Although prophylactic vaccination against SARS-CoV-2 infection has been shown to be effective, no definitive treatment exists to date in the event of infection. The rapid spread of infection by SARS-CoV-2 and its variants fully warrants the continued evaluation of drug treatments for COVID-19, especially in the context of repurposing of already available and safe drugs. Here, we explored the therapeutic potential of melatonin and melatonergic compounds in attenuating COVID-19 pathogenesis in mice expressing human ACE2 receptor (K18-hACE2), strongly susceptible to SARS-CoV-2 infection. Daily administration of melatonin, agomelatine, or ramelteon delays the occurrence of severe clinical outcome with improvement of survival, especially with high melatonin dose. Although no changes in most lung inflammatory cytokines are observed, treatment with melatonergic compounds limits the exacerbated local lung production of type I and type III interferons, which is likely associated with the observed improved symptoms in treated mice. The promising results from this preclinical study should encourage studies examining the benefits of repurposing melatonergic drugs to treat COVID-19 and related diseases in humans.

Use of candles and risk of cardiovascular and respiratory events in a Danish cohort study.

Burning candles at home emit small particles and gases that pollute indoor air. Exposure to fine particles in outdoor air has been convincingly linked to cardiovascular and respiratory events, while the associations with fine and ultrafine particles from candle burning remain unexplored. We examined the association between the use of candles and incident cardiovascular and respiratory events. We collected data on 6757 participants of the Copenhagen Aging and Midlife Biobank cohort recruited in 2009 and followed them up for the first hospital contact for incident cardiovascular and respiratory events until 2018. We investigated an association between the self-reported frequency of candle use in wintertime and cardiovascular and respiratory events, using Cox regression models adjusting for potential confounders. During follow-up, 1462 and 834 were admitted for cardiovascular and respiratory events, respectively. We found null associations between candle use and a hospital contact due to cardiovascular and respiratory events, with hazard ratios (HRs) and 95% confidence intervals (CI) of 0.97 (95% CI: 0.84, 1.11) and 0.98 (95% CI: 0.81, 1.18), respectively, among those using candles >4 times/week compared with <1 time/week. For cause-specific cardiovascular diseases, HRs were 1.10 (95% CI: 0.85, 1.43) for ischemic heart disease and 1.18 (95% CI: 0.77, 1.81) for myocardial infarction. For chronic obstructive pulmonary disease, HR was 1.26 (95% CI: 0.81, 1.97). We found no statistically significant associations between candle use and the risk of cardiovascular and respiratory events. Studies with improved exposure assessments are warranted.

Amyloid Beta Peptide Is an Endogenous Negative Allosteric Modulator of Leptin Receptor.

INTRODUCTION: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. OBJECTIVE: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aß) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. METHODS: We developed a new time-resolved fluorescence resonance energy transfer-based Aß binding assay for the leptin receptor (LepR) and studied the effect of Aß on LepR function in several in vitro assays. The in vivo effect of Aß on LepR function was studied in an Aß-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. RESULTS: We revealed specific and high-affinity (Ki = 0.1 nM) binding of Aß to LepR. Pharmacological characterization of this interaction showed that Aß binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aß was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitroand in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aß. CONCLUSION: Our data indicate that Aß is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aß oligomers. Preventing the interaction of Aß with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

Endospanin 1 Determines the Balance of Leptin-Regulated Hypothalamic Functions.

Endospanin 1 (Endo1), a protein encoded in humans by the same gene than the leptin receptor (ObR), and increased by diet-induced obesity, is an important regulator of ObR trafficking and cell surface exposure, determining leptin signaling strength. Defective intracellular trafficking of the leptin receptor to the neuronal plasma membrane has been proposed as a mechanism underlying the development of leptin resistance observed in human obesity. More recently, Endo1 has emerged as a mediator of "selective leptin resistance." The underlying mechanisms of the latter are not completely understood, but the possibility of differential activation of leptin signaling pathways was suggested among others. In this respect, the expression level of Endo1 is crucial for the appropriate balance between different leptin signaling pathways and leptin functions in the hypothalamus and is likely participating in selective leptin resistance for the control of energy and glucose homeostasis.

Nocturnal activation of melatonin receptor type 1 signaling modulates diurnal insulin sensitivity via regulation of PI3K activity.

Recent genetic studies have highlighted the potential involvement of melatonin receptor 1 (MT1 ) and melatonin receptor 2 (MT2 ) in the pathogenesis of type 2 diabetes. Here, we report that mice lacking MT1 (MT1 KO) tend to accumulate more fat mass than WT mice and exhibit marked systemic insulin resistance. Additional experiments revealed that the main insulin signaling pathway affected by the loss of MT1 was the activation of phosphatidylinositol-3-kinase (PI3K). Transcripts of both catalytic and regulatory subunits of PI3K were strongly downregulated within MT1 KO mice. Moreover, the suppression of nocturnal melatonin levels within WT mice, by exposing mice to constant light, resulted in impaired PI3K activity and insulin resistance during the day, similar to what was observed in MT1 KO mice. Inversely, administration of melatonin to WT mice exposed to constant light was sufficient and necessary to restore insulin-mediated PI3K activity and insulin sensitivity. Hence, our data demonstrate that the activation of MT1 signaling at night modulates insulin sensitivity during the day via the regulation of the PI3K transcription and activity. Lastly, we provide evidence that decreased expression of MTNR1A (MT1 ) in the liver of diabetic individuals is associated with poorly controlled diabetes.

Increased expression of fibroblast growth factor 21 (FGF21) during chronic undernutrition causes growth hormone insensitivity in chondrocytes by inducing leptin receptor overlapping transcript (LEPROT) and leptin receptor overlapping transcript-like 1 (LEPROTL1) expression.

During calorie restriction in mice, increased expression of FGF21 causes growth attenuation and growth hormone (GH) insensitivity. Previous evidence also indicates that fasting-associated increased expression of leptin receptor overlapping transcript (LEPROT) and LEPROT-like 1 (LEPROTL1) (two proteins that regulate intracellular protein trafficking) reduces GH receptor cell-surface expression in the liver. Thus, we hypothesized that FGF21 causes GH insensitivity through regulation of LEPROT and/or LEPROTL1 expression. After 4 weeks of food restriction, LEPROT and LEPROTL1 mRNA expression in the liver and in the tibial growth plate of wild-type (WT) mice was increased compared with WT mice fed ad libitum. In Fgf21 knock-out (KO) mice, LEPROT and LEPROTL1 mRNA expression in food-restricted and fed ad libitum was similar, with the exception of a subgroup of food-restricted Fgf21 KO mice treated with recombinant human (rh) FGF21 that experienced increased LEPROT and LEPROTL1 mRNA expression compared with untreated food-restricted Fgf21 KO mice. In cultured growth plate chondrocytes, FGF21 stimulated LEPROT and LEPROTL1 mRNA expression, with such effect being prevented in chondrocytes transfected with FGFR1 siRNA or ERK1 siRNA. In cells transfected with control siRNA, GH increased [(3)H]thymidine incorporation, collagen X, and IGF-1 mRNA expression, with all effects being prevented by rhFGF21. In addition, rhFGF21 decreased (125)I-GH binding. In LEPROT siRNA- and/or LEPROTL1 siRNA-transfected cells, rhFGF21 did not prevent the GH stimulatory effects on thymidine incorporation, collagen X, and IGF-1 expression; furthermore, rhFGF21 did not prevent (125)I-GH binding. Consistent with the effects of rhFGF21, LEPROT overexpression in chondrocytes resulted in the inhibition of GH action. Our findings indicate that the increased expression of FGF21 during chronic undernutrition inhibits GH action on chondrocytes by activating LEPROT and LEPROTL1.

ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system.

G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group 'Biological roles of signal transduction'. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them.

Whole-body deletion of Endospanin 1 protects from obesity-associated deleterious metabolic alterations.

The importance of the proper localization of most receptors at the cell surface is often underestimated, although this feature is essential for optimal receptor response. Endospanin 1 (Endo1) (also known as OBRGRP or LEPROT) is a protein generated from the same gene as the human leptin receptor and regulates the trafficking of proteins to the surface, including the leptin receptor. The systemic role of Endo1 on whole-body metabolism has not been studied so far. Here, we report that general Endo1-KO mice fed a high-fat diet develop metabolically healthy obesity with lipid repartitioning in organs and preferential accumulation of fat in adipose tissue, limited systematic inflammation, and better controlled glucose homeostasis. Mechanistically, Endo1 interacts with the lipid translocase CD36, thus regulating its surface abundance and lipid uptake in adipocytes. In humans, the level of Endo1 transcripts is increased in the adipose tissue of patients with obesity, but low levels rather correlate with a profile of metabolically healthy obesity. We suggest here that Endo1, most likely by controlling CD36 cell surface abundance and lipid uptake in adipocytes, dissociates obesity from diabetes and that its absence participates in metabolically healthy obesity.

A plastic aggrecan barrier modulated by peripheral energy state gates metabolic signal access to arcuate neurons.

The hypothalamic arcuate nucleus (ARH) contains neurons vital for maintaining energy homeostasis that sense and respond to changes in blood-borne metabolic hormones. Despite its juxtaposition to the median eminence (ME), a circumventricular organ lacking a blood-brain barrier and thus exposed to circulating molecules, only a few ventral ARH neurons perceive these extravasating metabolic signals due to a poorly understood ME/ARH diffusion barrier. Here, we show in male mice that aggrecan, a perineural-net proteoglycan deposited by orexigenic ARH neurons, creates a peculiar ventrodorsal diffusion gradient. Fasting enhances aggrecan deposition more dorsally, reinforcing the diffusion barrier, particularly around neurons adjacent to fenestrated capillary loops that enter the ARH. The disruption of aggrecan deposits results in unregulated diffusion of blood-borne molecules into the ARH and impairs food intake. Our findings reveal the molecular nature and plasticity of the ME/ARH diffusion barrier, and indicate its physiological role in hypothalamic metabolic hormone sensing.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.

Development of indolealkylamine derivatives as potential multi-target agents for COVID-19 treatment.

COVID-19 is a complex disease with short-term and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. As many drugs targeting single targets showed only limited effectiveness against COVID-19, here, we aimed to explore a multi-target strategy. We synthesized a focused compound library based on C2-substituted indolealkylamines (tryptamines and 5-hydroxytryptamines) with activity for three potential COVID-19-related proteins, namely melatonin receptors, calmodulin and human angiotensin converting enzyme 2 (hACE2). Two molecules from the library, 5e and h, exhibit affinities in the high nanomolar range for melatonin receptors, inhibit the calmodulin-dependent calmodulin kinase II activity and the interaction of the SARS-CoV-2 Spike protein with hACE2 at micromolar concentrations. Both compounds inhibit SARS-CoV-2 entry into host cells and 5h decreases SARS-CoV-2 replication and MPro enzyme activity in addition. In conclusion, we provide a proof-of-concept for the successful design of multi-target compounds based on the tryptamine scaffold. Optimization of these preliminary hit compounds could potentially provide drug candidates to treat COVID-19 and other coronavirus diseases.

Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells.

Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer's disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

A seeding-based neuronal model of tau aggregation for use in drug discovery.

Intracellular accumulation of tau protein is a hallmark of Alzheimer's Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies.

Human GLP1R variants affecting GLP1R cell surface expression are associated with impaired glucose control and increased adiposity.

The glucagon-like peptide 1 receptor (GLP1R) is a major drug target with several agonists being prescribed in individuals with type 2 diabetes and obesity1,2. The impact of genetic variability of GLP1R on receptor function and its association with metabolic traits are unclear with conflicting reports. Here, we show an unexpected diversity of phenotypes ranging from defective cell surface expression to complete or pathway-specific gain of function (GoF) and loss of function (LoF), after performing a functional profiling of 60 GLP1R variants across four signalling pathways. The defective insulin secretion of GLP1R LoF variants is rescued by allosteric GLP1R ligands or high concentrations of exendin-4/semaglutide in INS-1 823/3 cells. Genetic association studies in 200,000 participants from the UK Biobank show that impaired GLP1R cell surface expression contributes to poor glucose control and increased adiposity with increased glycated haemoglobin A1c and body mass index. This study defines impaired GLP1R cell surface expression as a risk factor for traits associated with type 2 diabetes and obesity and provides potential treatment options for GLP1R LoF variant carriers.

Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway.

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.