Biology subjects

Navarro, G.

Publications and source records attributed to Navarro, G..

11 recordsLinked to original sources

bioRxiv2025

Tau oligomers modulate synapse fate by eliciting progressive bipartite synapse dysregulation and synapse loss

BackgroundSynapse function is critical for cognition, and synapse loss is highly correlated with cognitive decline in Alzheimers disease and related dementias. Tau oligomers, which accumulate in the brain in Alzheimers disease, can acutely inhibit synaptic plasticity and cause synapse loss. Coordinated presynaptic and postsynaptic function is essential for effective synaptic transmission, and both compartments can be dysregulated by pathogenic tau. However, the series of pathophysiological events triggered by tau oligomers to cause the dysfunction and deterioration of presynaptic terminals and postsynaptic sites remain unclear. MethodsWe developed a proximity labeling tool to map the postsynaptic proteome by fusing PSD-95 with APEX2 (APEX2-PSD-95) which was expressed in human induced pluripotent stem cell (iPSC)-derived neurons. We used APEX2-PSD-95 to map the dynamic changes in the postsynaptic proteome with precise temporal resolution after an acute exposure of human iPSC-derived neurons to recombinant tau oligomers for 30 min. Leveraging immunocytochemistry, electrophysiology and electron microscopy, we further delineated the impact of the acute tau oligomer exposure on presynaptic and postsynaptic compartments over time for up to 14 days. ResultsThe brief exposure of human iPSC-derived neurons to tau oligomers caused a progressive deterioration of synapses, marked by both presynaptic and postsynaptic dysregulation. Postsynaptic proteome mapping revealed an immediate tau oligomer-triggered downregulation of the postsynaptic actin motor proteins Myosin-Va and Myosin-10, which coincided with impaired AMPA receptor (AMPAR) trafficking during synaptic plasticity. This was followed 24 hours later by the upregulation of disease-related proteins, including GSK3{beta}, at postsynaptic sites. The loss of PSD-95-labeled postsynaptic sites at 7 days after tau oligomer exposure preceded the loss of Synapsin-labeled presynaptic terminals at 14 days. The postsynaptic sites that remained exhibited a long-term downregulation of postsynaptic AMPAR levels and sustained synaptic plasticity impairment. Moreover, the remaining presynaptic terminals contained less clusters of vesicles at the presynaptic active zone which was associated with reduced vesicle release probability at synapses. ConclusionOur findings reveal the series of events underlying tau oligomer-induced bipartite synapse deterioration. The progressive decline of synapses involves the emergence of two synapse fates. One synapse fate involves the persistent weakening of both presynaptic and postsynaptic function, and the other results in synaptic loss.

neuroscience↗

bioRxiv2025

The interplay between CB2 and NMDA receptors in Parkinsons disease

Parkinsons disease (PD) is a progressive neurological disorder that affects movement, causing symptoms such as tremors, stiffness, slowness, and balance problems due to the degeneration of dopamine-producing neurons in the brain. Nowadays there is no cure for PD. Alpha synuclein (-syn) aggregates, which are a hallmark of PD, are known to induce microglial activation, specifically the detrimental M1 microglial phenotype, which contributes to neuroinflammation and disease progression. Cannabinoid receptor 2 (CB2R) activation has been shown to counteract neuroinflammation. CB2R is able to interact with NMDA receptors (NMDAR), which has also attracted attention in PD research due to its role in excitotoxicity. Here we aimed to study the interaction between CB2R and NMDAR in a PD context. We observed that -syn fibrils alter CB2R activation and CB2R-NMDAR heteromerization in a heterologous expression system. Furthermore, activation of CB2R counteracted NMDAR signaling. In neurons -syn fibrils decreased CB2R-NMDAR heteromer expression, while increasing CB2R signaling. Importantly, CB2R activation counteracted the -syn fibrils-induced increase in M1 activated microglia, while it favored the polarization of microglia to the beneficial M2 phenotype. These results reinforce the idea of using cannabinoids for treating PD, as they provide not only the anti-inflammatory effects of cannabinoids but also counteract the detrimental increase in NMDAR signaling present in this disease.

neuroscience↗

bioRxiv2024

Homodimerization of CB2 cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we have shown that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB2 receptor (CB2R) homodimerization by simultaneously binding both protomers of the CB2R-CB2R homodimer. Computational and pharmacological experimentals showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This provides unique pharmacological properties, such as increased potency in Gi binding and increased recruitment of {beta}-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB2R activity with potentially improved therapeutic outcomes. HIGHLIGHTSO_LIA homobivalent ligand of CB2R (PM369) modulates the dynamics of receptor homodimerization C_LIO_LIPM369 binds to the orthosteric site of one protomer and to a complementary, membrane-facing, site of the other protomer C_LIO_LIPM369 triggers CB2R homodimerization via the TM 1/7 interface that provides unique pharmacological properties C_LIO_LIPM369 potentiates signaling, increased potency in Gi binding and increased recruitment of {beta}-arrestin C_LIO_LIThese results highlight new approaches to control GPCR signaling C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/593612v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1736aeborg.highwire.dtl.DTLVardef@707654org.highwire.dtl.DTLVardef@168925borg.highwire.dtl.DTLVardef@60bc11_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

molecular biology↗

bioRxiv2023

Fluorescent Tools for Imaging and Ligand Screening of Dopamine D2-Like Receptors

The family of dopamine D2-like receptors represent an interesting target for a variety of neurological diseases, e.g. Parkinsons disease (PD), addiction or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2-like receptors (pKi (D2longR) = 8.24, pKi (D3R) = 8.58, pKi (D4R) = 7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligands binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups.

molecular biology↗

bioRxiv2021

Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis

Background and aimsNormal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM - maternal metabolism interaction. MethodsThe GM and plasma metabolome of CD1, NIH-Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT). ResultsSignificant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post- pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy. ConclusionsGM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

physiology↗

bioRxiv2020

Nr2e3 functional domain ablation by CRISPR-Cas9D10A identifies a new isoform and generates Retinitis Pigmentosa and Enhanced S-cone Syndrome models

Mutations in NR2E3 cause retinitis pigmentosa (RP) and enhanced S-cone syndrome (ESCS) in humans. This gene produces a large isoform encoded in 8 exons and a previously unreported shorter isoform of 7 exons, whose function is unknown. We generated two mouse models by targeting exon 8 of Nr2e3 using CRISPR/Cas9-D10A nickase. Allele {Delta}27 is an in-frame deletion of 27 bp that ablates the dimerization domain, whereas allele {Delta}E8 (full deletion of exon 8), produces only the short isoform that lacks the dimerization and repressor domains. The {Delta}27 mutant shows developmental alterations and a non-progressive electrophysiological dysfunction that resembles the ESCS phenotype. The {Delta}E8 mutant exhibits progressive retinal degeneration, as occurs in human RP patients. Interestingly, the mutant retinas show invaginations similar to fovea-like pits. Our mutants suggest a role of Nr2e3 as a cone-patterning regulator and provide valuable models for studying mechanisms of NR2E3-associated retinal dystrophies and evaluating potential therapies. O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/147785v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1f5916forg.highwire.dtl.DTLVardef@11f1384org.highwire.dtl.DTLVardef@13a6cd2org.highwire.dtl.DTLVardef@be52b3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Nr2e3 mouse models were generated by exon 8 deletion using CRISPR/Cas9 D10A nickase. - New Nr2e3 mRNA retaining intron 7 encodes a short protein expressed in adult retina. - Deletion of 9 aa of the NR2E3 dimerization domain causes enhanced S-cone syndrome. - Deletion of exon 8 produces a phenotype similar to Retinitis Pigmentosa in mouse.

genetics↗

bioRxiv2019

DIMERBOW: exploring possible GPCR dimer interfaces

G protein-coupled receptors (GPCRs) can form homo-, heterodimers and larger order oligomers that exert different functions than monomers. The pharmacological potential of such complexes is hampered by the limited information available on the type of complex formed and its quaternary structure. Several GPCR structures in the Protein Data Bank display crystallographic interfaces potentially compatible with physiological interactions. Here we present DIMERBOW, a database and web application aimed to visually browse the complete repertoire of potential GPCR dimers present in solved structures. The tool is suited to help finding the best possible structural template to model GPCR homomers. DIMERBOW is available at http://lmc.uab.es/dimerbow/.

bioinformatics↗

bioRxiv2019

Adrenergic-melatonin heteroreceptor complexes are key in controlling ion homeostasis and intraocular eye pressure and their disruption contributes to hypertensive glaucoma

Melatonin regulates intraocular pressure (IOP) whose increase leads to glaucoma and eye nerve degeneration. Aiming at elucidating the role of melatonin receptors in humour production and IOP maintenance, we here demonstrate that glaucoma correlates with disassembly of 1-adrenergic/melatonin receptor functional units in cells producing the aqueous humour. Remarkably, 1-adrenoceptor-containing complexes do not coupled to the cognate Gq protein and, hence, phenylephrine activation of these receptors does not lead to Ca2+ mobilization. Functional complexes are significantly decreased in models of glaucoma and, more importantly, in human samples of glaucoma patients (GP). In such glaucomatous conditions phenylephrine produces, via 1-adrenoceptor activation, an increase in cytoplasmic [Ca2+] that is detrimental in glaucoma. The results led to hypothesize that using melatonin, a hypotensive agent, plus blockade of 1-adrenergic receptors may normalize pressure in glaucoma. Remarkably, co-instillation of melatonin and prazosin, a 1-adrenergic receptor antagonist, results in long-term decreases in IOP in a well-established animal model of glaucoma. The findings are instrumental to understand the physiological function of melatonin in the eye and its potential to address eye pathologies by targeting melatonin receptors and their complexes.

physiology↗

bioRxiv2019

The Michaelis-Menten paradox: Km is not an equilibrium constant but a steady-state constant.

Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, therefore, substrate affinity measured as the inverse of ES {rightleftarrows} E+S dissociation equilibrium constant (Kd) has a doubtful physiological meaning; in practice it is almost impossible to determine Kd (except using stopped-flow or other sophisticated methodologies). The Michaelis-Menten constant (Km), the concentration of substrate ([S]) providing half of enzyme maximal activity, is not the (Kd). In the simple E+S {rightleftarrows} ES [->] E+P or in more complex models describing S conversion into P, Km must be considered the constant defining the steady state at any substrate concentration. Enzyme kinetics is based on initial rate determination, i.e. in the linear part of the S to P conversion when the concentration of [ES] remains constant while steady state occurs. We also show that Systems Biology issues such as the time required to respond to a system perturbation, is more dependent on k1, the kinetic constant defining substrateenzyme association, than on Km. Whereas Km is instrumental for biochemical basic and applied approaches, in any physiological condition, an important parameter to be considered is the substrate association rate (k1).

biochemistry↗

bioRxiv2018

Gut microbial features can predict host phenotype response to protein deficiency

Malnutrition remains a major health problem in low and middle income countries. During low protein intake, < 0.67 g/kg/day, there is a loss of nitrogen (N2) balance, due to the unavailability of amino acid for metabolism and unbalanced protein catabolism results. However, there are individuals, who consume the same low protein intake, and preserve N2 balance for unknown reasons. A novel factor, the gut microbiota, may account for these N2 balance differences. To investigate this, we correlated gut microbial profiles with the growth of four murine strains (C57Bl6/J, CD-1, FVB, and NIH-Swiss) on protein deficient (PD) diet. Results show that a PD diet exerts a strain-dependent impact on growth and N2 balance as determined through analysis of urinary urea, ammonia and creatinine excretion. Bacterial alpha diversity was significantly (p < 0.05, FDR) lower across all strains on a PD diet compared to normal chow (NC). Multi-group analyses of the composition of microbiomes (ANCOM) revealed significantly differential microbial signatures between the four strains independent of diet. However, mice on a PD diet demonstrated differential enrichment of bacterial genera including, Allobaculum (C57Bl6/J), Parabacteroides (CD-1), Turicibacter (FVB), and Mucispirillum (NIH-Swiss) relative to NC. Additionally, statistical model fitting revealed that the relative abundance of genera such as Bifidobacterium, Ruminococcus, and Lactobacillus were significantly positively correlated with body weight, while Anaerofustis, Roseburia, and Bilophila were significantly positively correlated with ammonia excretion. Taken together, these results suggest a potential relationship between the specific gut microbiota, N2 balance and animal response to malnutrition.

physiology↗