Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

All-optical physiology resolves a synaptic basis for behavioral timescale plasticity.

Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.

A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase ß subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

Plasticity of the Electrical Connectome of C. elegans.

The specific patterns and functional properties of electrical synapses of a nervous system are defined by the neuron-specific complement of electrical synapse constituents. We systematically examined the molecular composition of the electrical connectome of the nematode C. elegans through a genome- and nervous-system-wide analysis of the expression patterns of the invertebrate electrical synapse constituents, the innexins. We observe highly complex combinatorial expression patterns throughout the nervous system and found that these patterns change in a strikingly neuron-type-specific manner throughout the nervous system when animals enter an insulin-controlled diapause arrest stage under harsh environmental conditions, the dauer stage. By analyzing several individual synapses, we demonstrate that dauer-specific electrical synapse remodeling is responsible for specific aspects of the altered locomotory and chemosensory behavior of dauers. We describe an intersectional gene regulatory mechanism involving terminal selector and FoxO transcription factors mediating dynamic innexin expression plasticity in a neuron-type- and environment-specific manner.

Continual Learning in a Multi-Layer Network of an Electric Fish.

Distributing learning across multiple layers has proven extremely powerful in artificial neural networks. However, little is known about how multi-layer learning is implemented in the brain. Here, we provide an account of learning across multiple processing layers in the electrosensory lobe (ELL) of mormyrid fish and report how it solves problems well known from machine learning. Because the ELL operates and learns continuously, it must reconcile learning and signaling functions without switching its mode of operation. We show that this is accomplished through a functional compartmentalization within intermediate layer neurons in which inputs driving learning differentially affect dendritic and axonal spikes. We also find that connectivity based on learning rather than sensory response selectivity assures that plasticity at synapses onto intermediate-layer neurons is matched to the requirements of output neurons. The mechanisms we uncover have relevance to learning in the cerebellum, hippocampus, and cerebral cortex, as well as in artificial systems.

Spatially Stable Mitochondrial Compartments Fuel Local Translation during Plasticity.

Local translation meets protein turnover and plasticity demands at synapses, however, the location of its energy supply is unknown. We found that local translation in neurons is powered by mitochondria and not by glycolysis. Super-resolution microscopy revealed that dendritic mitochondria exist as stable compartments of single or multiple filaments. To test if these mitochondrial compartments can serve as local energy supply for synaptic translation, we stimulated individual synapses to induce morphological plasticity and visualized newly synthesized proteins. Depletion of local mitochondrial compartments abolished both the plasticity and the stimulus-induced synaptic translation. These mitochondrial compartments serve as spatially confined energy reserves, as local depletion of a mitochondrial compartment did not affect synaptic translation at remote spines. The length and stability of dendritic mitochondrial compartments and the spatial functional domain were altered by cytoskeletal disruption. These results indicate that cytoskeletally tethered local energy compartments exist in dendrites to fuel local translation during synaptic plasticity.

Distinct Dopamine Receptor Pathways Underlie the Temporal Sensitivity of Associative Learning.

Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. Here, we show that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, we show that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. Our results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment.

Clock-Generated Temporal Codes Determine Synaptic Plasticity to Control Sleep.

Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identify the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer.

The neuronal gene Arc is essential for long-lasting information storage in the mammalian brain, mediates various forms of synaptic plasticity, and has been implicated in neurodevelopmental disorders. However, little is known about Arc's molecular function and evolutionary origins. Here, we show that Arc self-assembles into virus-like capsids that encapsulate RNA. Endogenous Arc protein is released from neurons in extracellular vesicles that mediate the transfer of Arc mRNA into new target cells, where it can undergo activity-dependent translation. Purified Arc capsids are endocytosed and are able to transfer Arc mRNA into the cytoplasm of neurons. These results show that Arc exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis indicates that Arc is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses. These findings suggest that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system.

Regulation of Neuronal Differentiation, Function, and Plasticity by Alternative Splicing.

Posttranscriptional mechanisms provide powerful means to expand the coding power of genomes. In nervous systems, alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of transcripts. Thus, alternative splicing programs play instructive roles in the development of neuronal cell type-specific properties, neuronal growth, self-recognition, synapse specification, and neuronal network function. Here we discuss the most recent genome-wide efforts on mapping RNA codes and RNA-binding proteins for neuronal alternative splicing regulation. We illustrate how alternative splicing shapes key steps of neuronal development, neuronal maturation, and synaptic properties. Finally, we highlight efforts to dissect the spatiotemporal dynamics of alternative splicing and their potential contribution to neuronal plasticity and the mature nervous system.

Phosphorylation-dependent membraneless organelle fusion and fission illustrated by postsynaptic density assemblies.

Membraneless organelles formed by phase separation of proteins and nucleic acids play diverse cellular functions. Whether and, if yes, how membraneless organelles in ways analogous to membrane-based organelles also undergo regulated fusion and fission is unknown. Here, using a partially reconstituted mammalian postsynaptic density (PSD) condensate as a paradigm, we show that membraneless organelles can undergo phosphorylation-dependent fusion and fission. Without phosphorylation of the SAPAP guanylate kinase domain-binding repeats, the upper and lower layers of PSD protein mixtures form two immiscible sub-compartments in a phase-in-phase organization. Phosphorylation of SAPAP leads to fusion of the two sub-compartments into one condensate accompanied with an increased Stargazin density in the condensate. Dephosphorylation of SAPAP can reverse this event. Preventing SAPAP phosphorylation in vivo leads to increased separation of proteins from the lower and upper layers of PSD sub-compartments. Thus, analogous to membrane-based organelles, membraneless organelles can also undergo regulated fusion and fission.

Synaptic Mechanisms Regulating Mood State Transitions in Depression.

Depression is an episodic form of mental illness characterized by mood state transitions with poorly understood neurobiological mechanisms. Antidepressants reverse the effects of stress and depression on synapse function, enhancing neurotransmission, increasing plasticity, and generating new synapses in stress-sensitive brain regions. These properties are shared to varying degrees by all known antidepressants, suggesting that synaptic remodeling could play a key role in depression pathophysiology and antidepressant function. Still, it is unclear whether and precisely how synaptogenesis contributes to mood state transitions. Here, we review evidence supporting an emerging model in which depression is defined by a distinct brain state distributed across multiple stress-sensitive circuits, with neurons assuming altered functional properties, synapse configurations, and, importantly, a reduced capacity for plasticity and adaptation. Antidepressants act initially by facilitating plasticity and enabling a functional reconfiguration of this brain state. Subsequently, synaptogenesis plays a specific role in sustaining these changes over time.

A Functional Dissection of the mRNA and Locally Synthesized Protein Population in Neuronal Dendrites and Axons.

Neurons are characterized by a complex morphology that enables the generation of subcellular compartments with unique biochemical and biophysical properties, such as dendrites, axons, and synapses. To sustain these different compartments and carry a wide array of elaborate operations, neurons express a diverse repertoire of gene products. Extensive regulation at both the messenger RNA (mRNA) and protein levels allows for the differentiation of subcellular compartments as well as numerous forms of plasticity in response to variable stimuli. Among the multiple mechanisms that control cellular functions, mRNA translation is manipulated by neurons to regulate where and when a protein emerges. Interestingly, transcriptomic and translatomic profiles of both dendrites and axons have revealed that the mRNA population only partially predicts the local protein population and that this relation significantly varies between different gene groups. Here, we describe the space that local translation occupies within the large molecular and regulatory complexity of neurons, in contrast to other modes of regulation. We then discuss the specialized organization of mRNAs within different neuronal compartments, as revealed by profiles of the local transcriptome. Finally, we discuss the features and functional implications of both locally correlated-and anticorrelated-mRNA-protein relations both under baseline conditions and during synaptic plasticity.

RNA-binding proteins balance brain function in health and disease.

Posttranscriptional gene expression including splicing, RNA transport, translation, and RNA decay provides an important regulatory layer in many if not all molecular pathways. Research in the last decades has positioned RNA-binding proteins (RBPs) right in the center of posttranscriptional gene regulation. Here, we propose interdependent networks of RBPs to regulate complex pathways within the central nervous system (CNS). These are involved in multiple aspects of neuronal development and functioning, including higher cognition. Therefore, it is not sufficient to unravel the individual contribution of a single RBP and its consequences but rather to study and understand the tight interplay between different RBPs. In this review, we summarize recent findings in the field of RBP biology and discuss the complex interplay between different RBPs. Second, we emphasize the underlying dynamics within an RBP network and how this might regulate key processes such as neurogenesis, synaptic transmission, and synaptic plasticity. Importantly, we envision that dysfunction of specific RBPs could lead to perturbation within the RBP network. This would have direct and indirect (compensatory) effects in mRNA binding and translational control leading to global changes in cellular expression programs in general and in synaptic plasticity in particular. Therefore, we focus on RBP dysfunction and how this might cause neuropsychiatric and neurodegenerative disorders. Based on recent findings, we propose that alterations in the entire regulatory RBP network might account for phenotypic dysfunctions observed in complex diseases including neurodegeneration, epilepsy, and autism spectrum disorders.

The Genetic Control of Stoichiometry Underlying Autism.

Autism is a common and complex neurologic disorder whose scientific underpinnings have begun to be established in the past decade. The essence of this breakthrough has been a focus on families, where genetic analyses are strongest, versus large-scale, case-control studies. Autism genetics has progressed in parallel with technology, from analyses of copy number variation to whole-exome sequencing (WES) and whole-genome sequencing (WGS). Gene mutations causing complete loss of function account for perhaps one-third of cases, largely detected through WES. This limitation has increased interest in understanding the regulatory variants of genes that contribute in more subtle ways to the disorder. Strategies combining biochemical analysis of gene regulation, WGS analysis of the noncoding genome, and machine learning have begun to succeed. The emerging picture is that careful control of the amounts of transcription, mRNA, and proteins made by key brain genes-stoichiometry-plays a critical role in defining the clinical features of autism.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Synapse-type-specific competitive Hebbian learning forms functional recurrent networks.

Cortical networks exhibit complex stimulus-response patterns that are based on specific recurrent interactions between neurons. For example, the balance between excitatory and inhibitory currents has been identified as a central component of cortical computations. However, it remains unclear how the required synaptic connectivity can emerge in developing circuits where synapses between excitatory and inhibitory neurons are simultaneously plastic. Using theory and modeling, we propose that a wide range of cortical response properties can arise from a single plasticity paradigm that acts simultaneously at all excitatory and inhibitory connections-Hebbian learning that is stabilized by the synapse-type-specific competition for a limited supply of synaptic resources. In plastic recurrent circuits, this competition enables the formation and decorrelation of inhibition-balanced receptive fields. Networks develop an assembly structure with stronger synaptic connections between similarly tuned excitatory and inhibitory neurons and exhibit response normalization and orientation-specific center-surround suppression, reflecting the stimulus statistics during training. These results demonstrate how neurons can self-organize into functional networks and suggest an essential role for synapse-type-specific competitive learning in the development of cortical circuits.

The ISR downstream target ATF4 represses long-term memory in a cell type-specific manner.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.