Biology subjects

Senatore, A.

Publications and source records attributed to Senatore, A..

12 recordsLinked to original sources

bioRxiv2025

Functional and phylogenetic analysis of placozoan GPCRs reveal the prebilaterian origin of monoaminergic signalling.

Monoamines are biologically active compounds crucial for neurotransmission and various physiological processes. They include neurotransmitters like serotonin, dopamine, and melatonin, which regulate mood, movement, and sleep in humans. In ecdysozoans, monoamines such as tyramine are important for modulating locomotion, learning, and feeding. The monoaminergic signalling system has been considered a bilaterian innovation, with conflicting evidence supporting its existence in earlier branching, non-bilaterian animals. Here, we challenge the bilaterian origin hypothesis by combining large-scale receptor deorphanisation with phylogenetic analyses to identify monoamine receptors from the placozoan Trichoplax adhaerens. We demonstrate that these receptors are homologous to known bilaterian GPCRs, and behavioural assays demonstrate that monoamines like tyramine and tryptamine affect the speed of locomotion and body shape of this animal, respectively. These responses, together with the presence of biosynthetic enzymes for these molecules, reveal that monoaminergic signalling is both active and endogenous in placozoans. Our findings provide compelling evidence for a prebilaterian origin of monoaminergic systems, reshaping our understanding of early nervous system evolution.

evolutionary biology↗

bioRxiv2025

NALCN/Cch1 channelosome subunits originated in early eukaryotes and are fully conserved in animals, fungi, and apusomonads

The sodium leak channel NALCN, a key regulator of neuronal excitability, associates with three ancillary subunits that are critical for its function: an extracellular subunit called FAM155, and two cytoplasmic subunits called UNC79 and UNC80. Interestingly, NALCN and FAM155 have orthologous phylogenetic relationships with the fungal calcium channel Cch1 and its extracellular subunit Mid1, however, UNC79 and UNC80 have not been reported outside of animals. In this study, we leveraged expanded gene sequence data available for eukaryotes to re-examine the evolutionary origins of NALCN and Cch1 channel subunits. Our analysis corroborates the direct phylogenetic relationship between NALCN and Cch1 and identifies a larger clade of related channels in additional eukaryotic taxa. We also identify homologues of FAM155/Mid1 in Cryptista algae, and UNC79 and UNC80 homologues in numerous non-metazoan eukaryotes including basidiomycete and mucoromycete fungi, and the microbial eukaryotic taxa Apusomonadida, Malawimonadida, and Discoba. Furthermore, we find that most major animal lineages, except ctenophores, possess a full complement of NALCN subunits. Comparing structural predictions with the solved structure of the human NALCN complex supports orthologous relationships between metazoan and non-metazoan FAM155/Mid1, UNC79, and UNC80 homologues. Together, our analyses reveal unexpected diversity and ancient eukaryotic origins of NALCN/Cch1 channelosome subunits and raise interesting questions about the functional nature of this conserved channel complex within a broad, eukaryotic context.

evolutionary biology↗

bioRxiv2024

Evolution of iGluR ligand specificity, polyamine regulation, and ion selectivity inferred from a placozoan Epsilon receptor

Epsilon ionotropic glutamate receptors (iGluRs) belong to a recently described sub-family of metazoan receptors that is distinct from the AMPA, Kainate, Delta, and Phi (i.e., AKDF) sub-family, the NMDA sub-family, and the Lambda subfamily. Here, we sought to better understand the evolutionary and functional properties of Epsilon receptors by focusing on homologues from the basal invertebrate Trichoplax adhaerens (phylum Placozoa). We provide an updated species-guided phylogeny of eukaryotic iGluRs, and a comprehensive phylogeny of placozoan receptors uncovering marked diversification of Epsilon receptors within three conserved subclades, and four invariable subclades of AKDF receptors. Detailed functional characterization of the T. adhaerens Epsilon receptor GluE1A revealed robust activation by glycine, alanine, serine, and valine, but not glutamate. Through combined of structural modeling and mutation experiments, we used GluE1A to test the hypothesis that only a small set of amino acids in the ligand binding domain determine ligand selectivity. Mutation of just three amino acids converted GluE1A selectivity to glutamate, resulted in nascent sensitivity to AMPA, and increased sensitivity to the AMPA/Kainate receptor blocker CNQX. Lastly, combined modeling and mutation experiments revealed that an atypical serine residue in the pore NQR site of GluE1A, along with an aspartate four amino acids downstream, confers sensitivity to voltage-dependent polyamine block, while the serine alone diminishes both polyamine block and Ca2+ permeation compared to asparagine and glutamine residues of AMPA and Kainate receptors. Altogether, we demonstrate conserved molecular determinants for polyamine regulation between Epsilon and AKDF receptors, and evidence that natural variations in NQR residues have important implications for ion permeation and regulation by polyamines.

neuroscience↗

bioRxiv2024

The binding of Mint/X11 PDZ domains to CaV2 calcium channels predates bilaterian animals

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 channels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly binds the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates pre-bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.

evolutionary biology↗

bioRxiv2022

Function and phylogeny support the independent evolution of acid-sensing ion channels in the Placozoa

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are part of the Deg/ENaC ion channel family, which also includes neuropeptide-, bile acid-, and mechanically-gated channels. Despite sharing common tertiary and quaternary structures, strong sequence divergence within the Deg/ENaC family has made it difficult to resolve their phylogenetic relationships, and by extension, whether channels with common functional features, such as proton-activation, share common ancestry or evolved independently. Here, we report that a Deg/ENaC channel from the early diverging placozoan species Trichoplax adhaerens, named TadNaC2, conducts proton-activated currents in vitro with biophysical features that resemble those of the mammalian ASIC1 to ASIC3 channels. Through a combined cluster- based and phylogenetic analysis, we successfully resolve the evolutionary relationships of most major lineages of metazoan Deg/ENaC channels, identifying two subfamilies within the larger Deg/ENaC family that are of ancient, pre-bilaterian origin. We also identify bona fide Deg/ENaC channel homologues from filasterean and heterokont single celled eukaryotes. Furthermore, we find that ASIC channels, TadNaC2, and various other proton-activated channels from vertebrates and invertebrates are part of phylogenetically distinct lineages. Through structural modelling and mutation analysis, we find that TadNaC2 proton-activation employs fundamentally different molecular determinants than ASIC channels, and identify two unique histidine residues in the placozoan channel that are required for its proton-activation. Together, our phylogenetic and functional analyses support the independent evolution of proton-activated channels in the phylum Placozoa. Spurred by our discovery of pH sensitive channels, we discovered that despite lacking a nervous system, Trichoplax can sense changes in extracellular pH to coordinate its various cell types to locomote away from acidic environments, and to contract upon rapid exposure to acidic pH in a Ca2+-dependent manner. Lastly, via yeast 2 hybrid screening, we find that the Trichoplax channels TadNaC2 and TadNaC10, belonging to the two separate Deg/ENaC subfamilies, interact with the cytoskeleton organizing protein filamin, similar to the interaction reported for the human ENaC channels.

evolutionary biology↗

bioRxiv2022

Brain aging is faithfully modelled in organotypic brain slices and accelerated by prions

Mammalian models are essential for brain aging research. However, the long lifespan and limited amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in brain slices. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish a novel, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases.

neuroscience↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗

bioRxiv2021

Glial activation in prion diseases is strictly nonautonomous and requires neuronal PrPSc

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons, astrocytes or oligodendrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

neuroscience↗

bioRxiv2020

Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the peripheral demyelinating neuropathy was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands. Summary blurbA dimeric prion protein ligand activates Adgrg6 but fails to induce pro-myelination signaling upon chronic treatment in a mouse model of peripheral demyelination.

neuroscience↗

bioRxiv2020

Protective anti-prion antibodies in human immunoglobulin repertoires

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here we identified >6000 PrP-binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage-derived antibodies. When expressed recombinantly, these antibodies exhibited anti-PrP reactivity. Furthermore, we surveyed 48718 samples from 37894 hospital patients for the presence of anti-PrP IgGs, and found 21 high-titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti-PrP autoimmunity is innocuous. The existence of protective anti-prion antibodies in unbiased human immunological repertoires, combined with the reported lack of such antibodies in carriers of disease-associated PRNP mutations, suggests a link to the low incidence of spontaneous prion diseases in human populations.

neuroscience↗

bioRxiv2020

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3 interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing homologous domain architectures as known RIM homologues, but some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homologue. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

evolutionary biology↗

bioRxiv2017

Species and individual differences in serotonin receptor expression in identified neurons are correlated with a motor behavior

The marine mollusc, Pleurobranchaea californica varies daily in whether it swims and this correlates with whether serotonin (5-HT) enhances the strength of synapses made by the swim central pattern generator neuron, C2. Another species, Tritonia diomedea, reliably swims and does not vary in serotonergic neuromodulation. A third species, Hermissenda crassicornis, never produces this behavior and lacks the neuromodulation. We found that expression of particular 5-HT receptor genes in C2 correlates with swimming. Seven 5-HT receptor subtype genes were identified from whole-brain transcriptomes. We isolated individual C2 neurons and sequenced their RNA or measured 5-HT receptor gene expression using quantitative PCR. C2 neurons isolated from Pleurobranchaea individuals that produced a swim motor pattern just prior to isolation expressed the 5-HT2a and 5-HT7 receptor genes, as did the Tritonia samples. These subtypes were absent from C2 neurons isolated from Pleurobranchaea individuals that did not swim that day and from Hermissenda C2 neurons. Expression of other receptors did not correlate with swimming. This suggests that 5-HT2a and 5-HT7 receptors mediate the modulation of C2 synaptic strength and play an important role in swimming. Furthermore, the results suggest that regulation of receptor expression might underlie daily changes in behavior as well as behavioral evolution.

neuroscience↗