Biology subjects

Navedo, M. F.

Publications and source records attributed to Navedo, M. F..

9 recordsLinked to original sources

bioRxiv2026

Uncovering a Sex-Specific cAMP Landscape in Vascular Smooth Muscle

The second messenger, cyclic adenosine monophosphate (cAMP), plays a crucial role in regulating cellular function, including in vascular smooth muscle (VSM). While extensive research has focused on the compartmentalization of cAMP signaling in various cell types, the influence of biological sex on distinct cAMP pools and their functional implications in the VSM remains largely unexplored. To address this knowledge gap, we employed a multiscale experimental approach spanning targeted cAMP biosensors, wire myography, live-cell calcium imaging, proximity ligation assays, and in vivo ultrasound imaging. Our findings reveal a sex-specific distribution of cAMP signaling domains, with the cAMP pool selectively present in the SR of females but not males in the VSM. Ovariectomy abolished this sexual dimorphism, with SR cAMP pools disappearing in ovx females, mirroring the pattern observed in male cells. Males and ovx female VSM exhibited a significantly stronger RyR-PDE3/PDE4 association than sham females, suggesting that ovarian hormone-dependent sequestration of phosphodiesterases away from the SR licenses cAMP signaling in this compartment in female cells. Functionally, this sexually dimorphic cAMP compartmentalization correlates with smaller Ca2+ spark amplitude, reduced isoproterenol-induced relaxation, and higher pulse wave velocity in male and ovx compared to female VSM/vessels/mice. Together, these findings establish biological sex as a fundamental determinant of subcellular cAMP organization in VSM, with implications for understanding sex differences in vascular function and cardiovascular disease.

cell biology↗

bioRxiv2026

A Unifying Mechanism for Synaptic Amyloid beta Toxicity Beta Adrenergic Potentiation of the Ca2+ Channel CaV1.2 by Amyloid beta

Amyloid {beta} peptides (A{beta}) trigger Alzheimers disease (AD) but how has remained elusive. A{beta} stimulates the {beta}2 adrenergic receptor ({beta}2AR), which forms a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. LTCCs have been implicated in the etiology of dementia and AD. We show that A{beta} acutely potentiates CaV1.2 via the {beta}2AR, which triggers postsynaptic recruitment of Ca2+ permeable (CP) AMPARs in hippocampal cultures and impairs LTP in hippocampal slices within minutes. The long-term consequence is a loss of postsynaptic structure of glutamatergic synapses and neurotoxicity. Disrupting this signaling cascade with highly specific tools prevented all of these effects, unifying a number of currently divergent findings on A{beta} synaptotoxicity including dysregulation of AMPARs and synaptic plasticity. TEASERAmyloid {beta} peptide is the primary pathological agent in Alzheimers disease. It affects the nanoscale structure and function of glutamatergic synapses. The molecular mechanisms are largely unknown except for identification of several binding proteins including the {beta}2 adrenergic receptor. We show that this binding potently (EC50<100 nM) augments Ca2+ influx through the L-type Ca channel CaV1.2. This effect leads to improper recruitment of Ca2+-permeable glutamate receptors to postsynaptic sites (EC50<100 nM), synaptic dysfunction and ultimately neuronal death. This work identifies an essential mechanism in amyloid {beta} neurotoxicity and explains many of the observed postsynaptic alterations. HighlightsImmediate effects of A{beta}-induced stimulation of {beta}2AR on Cav1.2: O_LIA{beta} induces phosphorylation of Cav1.2 on S1928 by PKA C_LIO_LIA{beta} augments Cav1.2 activity via {beta}2AR-induced S1928 phosphorylation within seconds C_LI A{beta}-induced {beta}2AR - Cav1.2 signaling has the following synaptotoxic effects. O_LIA{beta} induces postsynaptic accumulation of Ca-permeable AMPARs via {beta}2AR - Cav1.2 signaling within 20 min C_LIO_LIA{beta} impairs long-term potentiation (LTP) via {beta}2AR - Cav1.2 signaling C_LIO_LIA{beta} impairs postsynaptic structure and neuronal viability over 24 h C_LIO_LIPotency of A{beta} in all the above effects is very high (100 nM A{beta} is saturating!) C_LIO_LIAll effects are prevented in S1928A KI mice and acute displaces {beta}2AR from Cav1.2 with tat-Pep1923 C_LI

neuroscience↗

bioRxiv2026

Intracellular Mechanosensation in Intestinal Smooth Muscle: Piezo1 Complexes Amplify Signaling Beyond the Surface

Mechanosensation is fundamentally viewed as a plasma membrane phenomenon. We challenge this paradigm by introducing intracellular mechanosensation in intestinal smooth muscle. We hypothesized that a distinct, organelle-based signaling axis exists to amplify mechanotransduction from the inside out. To test this, we investigated whether Piezo1, a canonical plasma membrane mechanosensor, also operates within the cell. Using tissue-level wire myography, high-resolution confocal microscopy, proximity ligation assays, and patch-clamp electrophysiology on freshly dissociated cells, we identified a functional intracellular signaling hub that starts at the sarcoplasmic reticulum (SR). Unlike surface transduction, this intracellular mechanism relies on a nanoscale multiprotein complex (<40 nm) comprising an SR sensor (intra-Piezo1) and an amplifier (Ryanodine Receptor, RyR), coupled with a PM effector (large-conductance, Ca2+-activated K+ channels, i.e., BKCa channels). Activating this intracellular complex generated massive BK-mediated outward currents independent of extracellular Ca{superscript 2} but strictly dependent on internal SR Ca{superscript 2} stores, confirming intrinsic organellar mechanotransduction. Within this complex, intra-Piezo1 and RyR are positioned to operate as a coupled SR Ca{superscript 2} release unit that activates BK channels at SR-PM junctions, driving potent membrane hyperpolarization that reduces smooth muscle contractility, revealing the intra-Piezo1 complex as a molecular brake on excitation. These findings demonstrate that mechanotransduction is not confined to the cell surface. Instead, a specialized Sensor-Amplifier-Effector complex originating at intracellular organelles amplifies cellular sensitivity to physical force, providing a critical gain-control system that restrains smooth muscle excitability and regulates GI motility. Key PointsO_LIIntracellular organelles contribute to mechanosensory signaling in GI smooth muscle cells, complementing plasma membrane mechanisms. C_LIO_LIIntra-Piezo1 form a nanoscale signaling complex (<40nm) on the sarcoplasmic reticulum (SR), linking the mechanosensor Piezo1 with RyR and large conductance, Ca2+-activated K+ channels. C_LIO_LIUnlike surface sensors, this intracellular complex functions via a "Sensor-Amplifier-Effector" mechanism in which intra-Piezo1 detects mechanical stress and triggers SR Ca2+ release, thereby activating a nearby RyR and large-conductance, Ca2+-activated K+ channel. C_LIO_LIEngaging this intracellular Piezo1-mediated axis significantly dampens smooth muscle contractility, identifying a critical gain-control system essential for regulating GI motility. C_LI

physiology↗

bioRxiv2022

α1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca2+ channel Cav1.2 activity and long-term potentiation in rodents

The cellular mechanisms mediating norepinephrine functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq- coupled 1-adrenergic receptors (ARs). 1AR signaling increased LTCC activity in hippocampal neurons. This regulation required PKC-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by 1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by norepinephrine. Indeed, a form of hippocampal LTP in young mice requires both the LTCC and 1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via 1AR - Pyk2 - Src signaling regulates synaptic strength.

neuroscience↗

bioRxiv2022

Half-calcified Calmodulin Promotes Basal Activity and Inactivation of the Calcium Channel CaV1.2.

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. We identified a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100 nM) that constitutively pre-associates with CaV1.2 to promote Po and CDI. We present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12, a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. The CaM12 N-lobe does not interact with the IQ motif. The CaM12 C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to wildtype CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI identifying Ca2/CaM as an important mediator of Ca signaling.

biochemistry↗

bioRxiv2021

Oligodendrocytes are a lifelong source of nuclear and ribosomal material for neurons in the mouse brain

Nuclear and ribosomal components define cell identity and function by regulating chromatin dynamics, gene expression, and protein turnover. Here we report that in the mouse central nervous system (CNS) under normal conditions, neurons accumulate nuclear and ribosomal material of oligodendrocyte (OL) origin. We show that neuronal accumulation of OL-derived nuclear and ribosomal material is brain area-specific, and in the cortex and hippocampal dentate gyrus gradually propagates during postnatal brain maturation. We further demonstrate that OL-to-neuron material transfer persists throughout adulthood and responds to neuroinflammation. We found that satellite OL of the gray matter form internuclear contacts with receiving neurons in the mouse brain. Similar close internuclear associations between satellite OL and neurons are present in the adult human cortex. Our findings provide the first evidence of wide-spread dynamic and selective OL-to-neuron nuclear and ribosomal material transfer in the mouse CNS and indicate that satellite OL serve as powerful mediators of neuronal function. Equivalent processes may occur in the human CNS and cause neurological disorders when dysregulated. One Sentence SummaryNeurons receive OL-derived nuclear and ribosomal material

neuroscience↗

bioRxiv2021

Genetically Engineered Strains for Combinatorial Cardiovascular Optobiology

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage -specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (opto1AR) and cAMP (opto{beta}2AR), or Ca2+- permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in HCN4BAC-CatCh2/MHC-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

physiology↗

bioRxiv2019

β-blockers augment L-type Ca2+ channel activity by target-selective spatially restricted β2AR-cAMP-PKA signaling in neurons

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report that some antagonists of {beta} adrenergic receptors ({beta}ARs) such as {beta}-blocker carvedilol and alprenolol activate {beta}2AR at nanomolar concentrations, which promote G protein signaling and cAMP/PKA activity without action of G protein receptor kinases (GRKs). The cAMP/PKA signal is restricted within the local plasma membrane domain, leading to selectively enhance PKA-dependent augment of endogenous L-type calcium channel (LTCC) activity but not AMPA receptor in hippocampal neurons. Moreover, we have engineered a mutant {beta}2AR that lacks serine 204 and 207 in the catecholamine binding pocket. This mutant can be preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant {beta}2AR in hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which {beta}-blocker-dependent activation of GPCRs at low ligand concentrations promotes local cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

cell biology↗

bioRxiv2019

Alpha-Actinin-1 promotes activity of the L-type Ca2+ Channel CaV1.2

The L-type Ca2+ channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of -actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to -actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 K1647A and Y1649A mutations, which impair -actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not -actinin-1 binding, decreased single channel open probability, gating charge movement, and its coupling to channel opening. Thus, -actinin recruits CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.

molecular biology↗