PolyQ length-based molecular encoding of vocalization frequency in FOXP2.

The transcription factor FOXP2, a regulator of vocalization- and speech/language-related phenotypes, contains two long polyQ repeats (Q1 and Q2) displaying marked, still enigmatic length variation across mammals. We found that the Q1/Q2 length ratio quantitatively encodes vocalization frequency ranges, from the infrasonic to the ultrasonic, displaying striking convergent evolution patterns. Thus, species emitting ultrasonic vocalizations converge with bats in having a low ratio, whereas species vocalizing in the low-frequency/infrasonic range converge with elephants and whales, which have higher ratios. Similar, taxon-specific patterns were observed for the FOXP2-related protein FOXP1. At the molecular level, we observed that the FOXP2 polyQ tracts form coiled coils, assembling into condensates and fibrils, and drive liquid-liquid phase separation (LLPS). By integrating evolutionary and molecular analyses, we found that polyQ length variation related to vocalization frequency impacts FOXP2 structure, LLPS, and transcriptional activity, thus defining a novel form of polyQ length-based molecular encoding of vocalization frequency.

Micro- and Nanoplastics' Effects on Protein Folding and Amyloidosis.

A significant portion of the world's plastic is not properly disposed of and, through various processes, is degraded into microscopic particles termed micro- and nanoplastics. Marine and terrestrial faunae, including humans, inevitably get in contact and may inhale and ingest these microscopic plastics which can deposit throughout the body, potentially altering cellular and molecular functions in the nervous and other systems. For instance, at the cellular level, studies in animal models have shown that plastic particles can cross the blood-brain barrier and interact with neurons, and thus affect cognition. At the molecular level, plastics may specifically influence the folding of proteins, induce the formation of aberrant amyloid proteins, and therefore potentially trigger the development of systemic and local amyloidosis. In this review, we discuss the general issue of plastic micro- and nanoparticle generation, with a focus on their effects on protein folding, misfolding, and their possible clinical implications.

Heptad stereotypy, S/Q layering, and remote origin of the SARS-CoV-2 fusion core.

The fusion of the SARS-CoV-2 virus with cells, a key event in the pathogenesis of Covid-19, depends on the assembly of a six-helix fusion core (FC) formed by portions of the spike protein heptad repeats (HRs) 1 and 2. Despite the critical role in regulating infectivity, its distinctive features, origin, and evolution are scarcely understood. Thus, we undertook a structure-guided positional and compositional analysis of the SARS-CoV-2 FC, in comparison with FCs of related viruses, tracing its origin and ongoing evolution. We found that clustered amino acid substitutions within HR1, distinguishing SARS-CoV-2 from SARS-CoV-1, enhance local heptad stereotypy and increase sharply the FC serine-to-glutamine (S/Q) ratio, determining a neat alternate layering of S-rich and Q-rich subdomains along the post-fusion structure. Strikingly, SARS-CoV-2 ranks among viruses with the highest FC S/Q ratio, together with highly syncytiogenic respiratory pathogens (RSV, NDV), whereas MERS-Cov, HIV, and Ebola viruses display low ratios, and this feature reflects onto S/Q segregation and H-bonding patterns. Our evolutionary analyses revealed that the SARS-CoV-2 FC occurs in other SARS-CoV-1-like Sarbecoviruses identified since 2005 in Hong Kong and adjacent regions, tracing its origin to >50 years ago with a recombination-driven spread. Finally, current mutational trends show that the FC is varying especially in the FC1 evolutionary hotspot. These findings establish a novel analytical framework illuminating the sequence/structure evolution of the SARS-CoV-2 FC, tracing its long history within Sarbecoviruses, and may help rationalize the evolution of the fusion machinery in emerging pathogens and the design of novel therapeutic fusion inhibitors.

PolyQ length co-evolution in neural proteins.

Intermolecular co-evolution optimizes physiological performance in functionally related proteins, ultimately increasing molecular co-adaptation and evolutionary fitness. Polyglutamine (polyQ) repeats, which are over-represented in nervous system-related proteins, are increasingly recognized as length-dependent regulators of protein function and interactions, and their length variation contributes to intraspecific phenotypic variability and interspecific divergence. However, it is unclear whether polyQ repeat lengths evolve independently in each protein or rather co-evolve across functionally related protein pairs and networks, as in an integrated regulatory system. To address this issue, we investigated here the length evolution and co-evolution of polyQ repeats in clusters of functionally related and physically interacting neural proteins in Primates. We observed function-/disease-related polyQ repeat enrichment and evolutionary hypervariability in specific neural protein clusters, particularly in the neurocognitive and neuropsychiatric domains. Notably, these analyses detected extensive patterns of intermolecular polyQ length co-evolution in pairs and clusters of functionally related, physically interacting proteins. Moreover, they revealed both direct and inverse polyQ length co-variation in protein pairs, together with complex patterns of coordinated repeat variation in entire polyQ protein sets. These findings uncover a whole system of co-evolving polyQ repeats in neural proteins with direct implications for understanding polyQ-dependent phenotypic variability, neurocognitive evolution and neuropsychiatric disease pathogenesis.

Compound Dynamics and Combinatorial Patterns of Amino Acid Repeats Encode a System of Evolutionary and Developmental Markers.

Homopolymeric amino acid repeats (AARs) like polyalanine (polyA) and polyglutamine (polyQ) in some developmental proteins (DPs) regulate certain aspects of organismal morphology and behavior, suggesting an evolutionary role for AARs as developmental "tuning knobs." It is still unclear, however, whether these are occasional protein-specific phenomena or hints at the existence of a whole AAR-based regulatory system in DPs. Using novel approaches to trace their functional and evolutionary history, we find quantitative evidence supporting a generalized, combinatorial role of AARs in developmental processes with evolutionary implications. We observe nonrandom AAR distributions and combinations in HOX and other DPs, as well as in their interactomes, defining elements of a proteome-wide combinatorial functional code whereby different AARs and their combinations appear preferentially in proteins involved in the development of specific organs/systems. Such functional associations can be either static or display detectable evolutionary dynamics. These findings suggest that progressive changes in AAR occurrence/combination, by altering embryonic development, may have contributed to taxonomic divergence, leaving detectable traces in the evolutionary history of proteomes. Consistent with this hypothesis, we find that the evolutionary trajectories of the 20 AARs in eukaryotic proteomes are highly interrelated and their individual or compound dynamics can sharply mark taxonomic boundaries, or display clock-like trends, carrying overall a strong phylogenetic signal. These findings provide quantitative evidence and an interpretive framework outlining a combinatorial system of AARs whose compound dynamics mark at the same time DP functions and evolutionary transitions.

Polyserine repeats promote coiled coil-mediated fibril formation and length-dependent protein aggregation.

Short polyserine (polyS) repeats are frequently found in proteins and longer ones are produced in neurological disorders such as Huntington disease (HD) owing to translational frameshifting or non-ATG-dependent translation, together with polyglutamine (polyQ) and polyalanine (polyA) repeats, forming intracellular aggregates. However, the physiological and pathological structures of polyS repeats are not clearly understood. Early studies highlighted their structural versatility, similar to other homopolymers whose conformation is influenced by the surrounding protein context. As polyS stretches are frequently near polyQ and polyA repeats, which can be part of coiled coil (CC) structures, and the frameshift-derived polyS repeats in HD directly flank CC heptads important for aggregation, we investigate here the structural and aggregation properties of polyS in the context of CC structures. We have taken advantage of peptide models, previously used to study polyQ and polyA in CCs, in which we inserted polyS repeats of variable length and studied them in comparison with polyQ and polyA peptides. We found that polyS repeats promote CC-mediated polymerization and fibrillization as revealed by circular dichroism, chemical crosslinking, and atomic force microscopy. Furthermore, they promote CC-based, length-dependent intracellular aggregation, which is negligible with 7 and widespread with 49 serines. These findings show that polyS repeats can participate in the formation of CCs, as previously found for polyQ and polyA, conferring to peptides distinctive structural properties with aggregation kinetics that are intermediate between those of polyA and polyQ CCs, and contribute to an overall structural definition of the pathophysiogical roles of homopolymeric repeats in CC structures.

MicroRNA-22 Gates Long-Term Heterosynaptic Plasticity in Aplysia through Presynaptic Regulation of CPEB and Downstream Targets.

The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping the interaction networks of the human proteome, and define proteome-wide knowledge that may guide the informed biological exploration of the role of AARs in protein interactions.

Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone ß-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases.

Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins.

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming ß sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.

Helix neuronal ensembles with controlled cell type composition and placement develop functional polysynaptic circuits on Micro-Electrode Arrays.

Neuronal cell cultures on Micro-Electrode Arrays (MEAs) provide an essential experimental tool for studying the connectivity and long-term activity of complex neuronal networks. MEA studies are generally based on the analysis of mixed neuronal populations constituted by a large number of cultured cells with cell type composition and connectivity patterns which are quite unpredictable a priori. In this work, we propose a different approach which consists of assembling on MEAs neuronal circuits formed by individually identifiable C1, C3, and B2 Helix neurons. Cells were plated under conditions of controlled number and position to form neuronal networks of defined composition. We performed multi-site electrophysiological recordings, and we characterized the firing dynamics. By means of cross-correlation analysis, we studied the electrophysiological properties of MEA-coupled microcircuits and characterized their activity patterns. We showed how the synaptic connectivity, actually observed in polysynaptic circuits of C1, C3 and B2 neurons, correlates well with the expected connectivity of C1-B2, B2-B2 and B2-C3 cell pairs as previously reported in conventional electrophysiological studies in culture.

Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a role for miR-124 in long-term plasticity of synapses in the mature nervous system.

F3/contactin-related proteins in Helix pomatia nervous tissue (HCRPs): distribution and function in neurite growth and neurotransmitter release.

By using antibodies against mouse F3/contactin, we found immunologically related glycoproteins expressed in the nervous tissue of the snail Helix pomatia. Helix contactin-related proteins (HCRPs) include different molecules ranging in size from 90 to 240 kD. Clones isolated from a cDNA expression library allowed us to demonstrate that these proteins are translated from a unique 6.3-kb mRNA, suggesting that their heterogeneity depends on posttranslational processing. This is supported by the results of endoglycosidase F treatment, which indicate that the high-molecular-weight components are glycosylation variants of the 90-kD chain. In vivo and in cultures, HCRPs antibodies label neuronal soma and neurite extensions, giving the appearance of both cytoplasmic and cell surface immunostaining. On the other hand, no expression is found on nonneural tissues. Functionally, HCRPs are involved in neurite growth control and appear to modulate neurotransmitter release, as indicated by the inhibiting effects of specific antibodies on both functions. These data allow the definition of HCRPs glycoproteins as growth-promoting molecules, suggesting that they play a role in neurite development and presynaptic terminal maturation in the invertebrate nervous system.

Phosphorylation of synapsin domain A is required for post-tetanic potentiation.

Post-tetanic potentiation (PTP) is a form of homosynaptic plasticity important for information processing and short-term memory in the nervous system. The synapsins, a family of synaptic vesicle (SV)-associated phosphoproteins, have been implicated in PTP. Although several synapsin functions are known to be regulated by phosphorylation by multiple protein kinases, the role of individual phosphorylation sites in synaptic plasticity is poorly understood. All the synapsins share a phosphorylation site in the N-terminal domain A (site 1) that regulates neurite elongation and SV mobilization. Here, we have examined the role of phosphorylation of synapsin domain A in PTP and other forms of short-term synaptic enhancement (STE) at synapses between cultured Helix pomatia neurons. To this aim, we cloned H. pomatia synapsin (helSyn) and overexpressed GFP-tagged wild-type helSyn or site-1-mutant helSyn mutated in the presynaptic compartment of C1-B2 synapses. We found that PTP at these synapses depends both on Ca2+/calmodulin-dependent and cAMP-dependent protein kinases, and that overexpression of the non-phosphorylatable helSyn mutant, but not wild-type helSyn, specifically impairs PTP, while not altering facilitation and augmentation. Our findings show that phosphorylation of site 1 has a prominent role in the expression of PTP, thus defining a novel role for phosphorylation of synapsin domain A in short-term homosynaptic plasticity.

Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons.

Synapsins are synaptic vesicle-associated phosphoproteins involved in the regulation of neurotransmitter release and synapse formation; they are substrates for multiple protein kinases that phosphorylate them on distinct sites. We have previously found that injection of synapsin into Helix snail neurons cultured under low-release conditions increases the efficiency of neurotransmitter release. In order to investigate the role of phosphorylation in this modulatory action of synapsins, we examined the substrate properties of the snail synapsin orthologue recently cloned in Aplysia (apSyn) for various protein kinases and compared the effects of the intracellular injection of wild-type apSyn with those of its phosphorylation site mutants. ApSyn was found to be an excellent in vitro substrate for cAMP-dependent protein kinase, which phosphorylated it at high stoichiometry on a single site (Ser-9) in the highly conserved domain A, unlike the other kinases reported to phosphorylate mammalian synapsins, which phosphorylated apSyn to a much lesser extent. The functional effect of apSyn phosphorylation by cAMP-dependent protein kinase on neurotransmitter release was studied by injecting wild-type or Ser-9 mutated apSyn into the soma of Helix serotonergic C1 neurons cultured under low-release conditions, i.e. in contact with the non-physiological target neuron C3. In this model of impaired neurotransmitter release, the injection of wild-type apSyn induced a significant enhancement of release. This enhancement was virtually absent after injection of the non-phosphorylatable mutant (Ser-9-->Ala), but it was maintained after injection of the pseudophosphorylated mutant (Ser-9-->Asp). These functional effects of apSyn injection were paralleled by marked ultrastructural changes in the C1 neuron, with the formation of extensive interdigitations of neurite-like processes containing an increased complement of C1 dense core vesicles at the sites of cell-to-cell contact. This structural rearrangement was virtually absent in mock-injected C1 neurons or after injection of the non-phosphorylatable apSyn mutant. These data indicate that phosphorylation of synapsin domain A is essential for the synapsin-induced enhancement of neurotransmitter release and suggest that endogenous kinases phosphorylating this domain play a central role in the regulation of the efficiency of the exocytotic machinery.

Inhibition of neurotransmitter release by a nonphysiological target requires protein synthesis and involves cAMP-dependent and mitogen-activated protein kinases.

During the development of neuronal circuits, axonal growth cones can contact many inappropriate targets before they reach an appropriate postsynaptic partner. Although it is well known that the contact with synaptic partners upregulates the secretory machinery of the presynaptic neuron, little is known about the signaling mechanisms involved in preventing the formation of connections with inappropriate target cells. Here, we show that the contact with a nonphysiological postsynaptic target inhibits neurotransmitter release from axonal terminals of the Helix serotonergic neuron C1 by means of an active mechanism requiring ongoing protein synthesis and leading to the inhibition of cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (Erk) pathways. The reversal of the inhibitory effect of the nonphysiological target by blockade of protein synthesis was prevented by cAMP-PKA or MAPK-Erk inhibitors, whereas disinhibition of neurotransmitter release promoted by cAMP-PKA activation was not affected by MAPK-Erk inhibitors. The data indicate that the inhibitory effect of the nonphysiological target on neurotransmitter release is an active process that requires protein synthesis and involves the downregulation of the MAPK-Erk and cAMP-PKA pathways, the same protein kinases that are activated after contact with a physiological target neuron. These mechanisms could play a relevant role in the prevention of synapse formation between inappropriate partners by modulating the neurotransmitter release capability of growing nerve terminals according to the nature of the targets contacted during their development.

Distribution of sensorin immunoreactivity in the central nervous system of Helix pomatia: functional aspects.

Land snails belonging to the genus Helix are commonly used to study several behaviors and their plasticity at the cellular level. Because the knowledge of sensory neurons in these species is far from being complete, we have investigated the presence and distribution in Helix pomatia central nervous system of the immunoreactivity for sensorin, a peptide specific for mechanosensory neurons in Aplysia. We found that the majority of immunopositive cells were grouped in clusters located in all the central ganglia, except for the pedal ganglion, where only a single large neuron was stained. A symmetrical cluster of stained cells in the cerebral ganglia showed homology with the cerebral J clusters in Aplysia. Most of the somata of these Helix cerebral clusters send their axons in the ipsilateral cerebropedal connective and lip nerves and make monosynaptic connections with cells located in a medial adjacent cluster. This monosynaptic circuit can be reestablished in culture, where it shows homosynaptic depression as it does in the ganglionic preparation.