ZAP facilitates NLRP3 inflammasome activation via promoting the oligomerization of NLRP3.

The NOD-like receptor family protein 3 (NLRP3) inflammasome is a crucial complex for the host to establish inflammatory immune responses and plays vital roles in a series of disorders, including Alzheimer's disease and acute peritonitis. However, its regulatory mechanism remains largely unclear. Zinc finger antiviral protein (ZAP), also known as zinc finger CCCH-type antiviral protein 1 (ZC3HAV1), promotes viral RNA degradation and plays vital roles in host antiviral immune responses. However, the role of ZAP in inflammation, especially in NLRP3 activation, is unclear. Here, we show that ZAP interacts with NLRP3 and promotes NLRP3 oligomerization, thus facilitating NLRP3 inflammasome activation in peritoneal macrophages of C57BL/6 mice. The shorter isoform of ZAP (ZAPS) appears to play a greater role than the full-length isoform (ZAPL) in HEK293T cells. Congruously, Zap-deficient C57BL/6 mice may be less susceptible to alum-induced peritonitis and lipopolysaccharide-induced sepsis in vivo. Therefore, we propose that ZAP is a positive regulator of NLRP3 activation and a potential therapeutic target for NLRP3-related inflammatory disorders.

An EEG Signature of MCH Neuron Activities Predicts Cocaine Seeking.

Background: Identifying biomarkers that predict substance use disorder (SUD) propensity may better strategize anti-addiction treatment. The melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) critically mediates interactions between sleep and substance use; however, their activities are largely obscured in surface electroencephalogram (EEG) measures, hindering the development of biomarkers. Methods: Surface EEG signals and real-time Ca2+ activities of LH MCH neurons (Ca2+MCH) were simultaneously recorded in male and female adult rats. Mathematical modeling and machine learning were then applied to predict Ca2+MCH using EEG derivatives. The robustness of the predictions was tested across sex and treatment conditions. Finally, features extracted from the EEG-predicted Ca2+MCH either before or after cocaine experience were used to predict future drug-seeking behaviors. Results: An EEG waveform derivative - a modified theta-to-delta ratio (EEG Ratio) - accurately tracks real-time Ca2+MCH in rats. The prediction was robust during rapid eye movement sleep (REMS), persisted through REMS manipulations, wakefulness, circadian phases, and was consistent across sex. Moreover, cocaine self-administration and long-term withdrawal altered EEG Ratio suggesting shortening and circadian redistribution of synchronous MCH neuron activities. In addition, features of EEG Ratio indicative of prolonged synchronous MCH neuron activities predicted lower subsequent cocaine seeking. EEG Ratio also exhibited advantages over conventional REMS measures for the predictions. Conclusions: The identified EEG Ratio may serve as a non-invasive measure for assessing MCH neuron activities in vivo and evaluating REMS; it may also serve as a potential biomarker predicting drug use propensity.

An Electroencephalogram Signature of Melanin-Concentrating Hormone Neuron Activities Predicts Cocaine Seeking.

BACKGROUND: Identifying biomarkers that predict substance use disorder propensity may better strategize antiaddiction treatment. Melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus critically mediate interactions between sleep and substance use; however, their activities are largely obscured in surface electroencephalogram (EEG) measures, hindering the development of biomarkers. METHODS: Surface EEG signals and real-time calcium (Ca2+) activities of lateral hypothalamus MCH neurons (Ca2+MCH) were simultaneously recorded in male and female adult rats. Mathematical modeling and machine learning were then applied to predict Ca2+MCH using EEG derivatives. The robustness of the predictions was tested across sex and treatment conditions. Finally, features extracted from the EEG-predicted Ca2+MCH either before or after cocaine experience were used to predict future drug-seeking behaviors. RESULTS: An EEG waveform derivative-a modified theta-delta-theta peak ratio (EEGTDT ratio)-accurately tracked real-time Ca2+MCH in rats. The prediction was robust during rapid eye movement sleep (REMS), persisted through vigilance states, sleep manipulations, and circadian phases, and was consistent across sex. Moreover, cocaine self-administration and long-term withdrawal altered EEGTDT ratio, suggesting shortening and circadian redistribution of synchronous MCH neuron activities. In addition, features of EEGTDT ratio indicative of prolonged synchronous MCH neuron activities predicted lower subsequent cocaine seeking. EEGTDT ratio also exhibited advantages over conventional REMS measures for the predictions. CONCLUSIONS: The identified EEGTDT ratio may serve as a noninvasive measure for assessing MCH neuron activities in vivo and evaluating REMS; it may also serve as a potential biomarker for predicting drug use propensity.

A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

The UAF1-USP1 Deubiquitinase Complex Stabilizes cGAS and Facilitates Antiviral Responses.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects cytoplasmic microbial DNA and self-DNA from genomic instability, initiates innate immunity, and plays fundamental roles in defense against viruses and the development of various diseases. The cellular cGAS level determines the magnitude of the response to DNA. However, the underlying mechanisms of the control of cGAS stability, especially its feedback regulation during viral infection, remain largely unknown. In this study, we show that viral infection induces the expression of the UAF1-USP1 deubiquitinase complex in primary peritoneal macrophages (PMs) of C57BL/6J mice. UAF1-USP interacts with cGAS, selectively cleaves its K48-linked polyubiquitination, and thus stabilizes its protein expression in PMs and HEK293T cells. Concordantly, the UAF1-USP1 deubiquitinase complex enhances cGAS-dependent type I IFN responses in PMs. Uaf1 deficiency and ML323 (a specific inhibitor of UAF1-USP1 deubiquitinase complex) attenuates cGAS-triggered antiviral responses and facilitates viral replication both in vitro and in vivo. Thus, our study uncovers a positive feedback mechanism of cGAS-dependent antiviral responses and suggests the UAF1-USP1 complex as a potential target for the treatment of diseases caused by aberrant cGAS activation.

Antagonistic circuits mediating infanticide and maternal care in female mice.

In many species, including mice, female animals show markedly different pup-directed behaviours based on their reproductive state1,2. Naive wild female mice often kill pups, while lactating female mice are dedicated to pup caring3,4. The neural mechanisms that mediate infanticide and its switch to maternal behaviours during motherhood remain unclear. Here, on the basis of the hypothesis that maternal and infanticidal behaviours are supported by distinct and competing neural circuits5,6, we use the medial preoptic area (MPOA), a key site for maternal behaviours7-11, as a starting point and identify three MPOA-connected brain regions that drive differential negative pup-directed behaviours. Functional manipulation and in vivo recording reveal that oestrogen receptor α (ESR1)-expressing cells in the principal nucleus of the bed nucleus of stria terminalis (BNSTprESR1) are necessary, sufficient and naturally activated during infanticide in female mice. MPOAESR1 and BNSTprESR1 neurons form reciprocal inhibition to control the balance between positive and negative infant-directed behaviours. During motherhood, MPOAESR1 and BNSTprESR1 cells change their excitability in opposite directions, supporting a marked switch of female behaviours towards the young.

A hypothalamic pathway that suppresses aggression toward superior opponents.

Aggression is costly and requires tight regulation. Here we identify the projection from estrogen receptor alpha-expressing cells in the caudal part of the medial preoptic area (cMPOAEsr1) to the ventrolateral part of the ventromedial hypothalamus (VMHvl) as an essential pathway for modulating aggression in male mice. cMPOAEsr1 cells increase activity mainly during male-male interaction, which differs from the female-biased response pattern of rostral MPOAEsr1 (rMPOAEsr1) cells. Notably, cMPOAEsr1 cell responses to male opponents correlated with the opponents' fighting capability, which mice could estimate based on physical traits or learn through physical combats. Inactivating the cMPOAEsr1-VMHvl pathway increased aggression, whereas activating the pathway suppressed natural intermale aggression. Thus, cMPOAEsr1 is a key population for encoding opponents' fighting capability-information that could be used to prevent animals from engaging in disadvantageous conflicts with superior opponents by suppressing the activity of VMHvl cells essential for attack behaviors.

Paraventricular Thalamus Dynamically Modulates Aversive Memory via Tuning Prefrontal Inhibitory Circuitry.

The impact of stress on the formation and expression of memory is well studied, especially on the contributions of stress hormones. But how stress affects brain circuitry dynamically to modulate memory is far less understood. Here, we used male C57BL6/J mice in an auditory fear conditioning as a model system to examine this question and focused on the impact of stress on dorsomedial prefrontal cortex (dmPFC) neurons which play an important role in probabilistic fear memory. We found that paraventricular thalamus (PVT) neurons are robustly activated by acute restraining stress. Elevated PVT activity during probabilistic fear memory expression increases spiking in the dmPFC somatostatin neurons which in turn suppresses spiking of dmPFC parvalbumin (PV) neurons, and reverts the usual low fear responses associated with probabilistic fear memory to high fear. This dynamic and reversible modulation allows the original memory to be preserved and modulated during memory expression. In contrast, elevated PVT activity during fear conditioning impairs synaptic modifications in the dmPFC PV-neurons and abolishes the formation of probabilistic fear memory. Thus, PVT functions as a stress sensor to modulate the formation and expression of aversive memory by tuning inhibitory functions in the prefrontal circuitry.SIGNIFICANCE STATEMENT The impact of stress on cognitive functions, such as memory and executive functions, are well documented especially on the impact by stress hormone. However, the contributions of brain circuitry are far less understood. Here, we show that a circuitry-based mechanism can dynamically modulate memory formation and expression, namely, higher stress-induced activity in paraventricular thalamus (PVT) impairs the formation and expression of probabilistic fear memory by elevating the activity of somatostatin-neurons to suppress spiking in dorsomedial prefrontal parvalbumin (PV) neurons. This stress impact on memory via dynamic tuning of prefrontal inhibition preserves the formed memory but enables a dynamic expression of memory. These findings have implications for better stress coping strategies as well as treatment options including better drug targets/mechanisms.

Rapid Eye Movement Sleep Engages Melanin-Concentrating Hormone Neurons to Reduce Cocaine Seeking.

BACKGROUND: Persistent sleep disruptions following withdrawal from abused drugs may hold keys to battle drug relapse. It is posited that there may be sleep signatures that predict relapse propensity, identifying which may open new avenues for treating substance use disorders. METHODS: We trained male rats (approximately postnatal day 56) to self-administer cocaine. After long-term drug withdrawal (approximately postnatal day 100), we examined the correlations between the intensity of cocaine seeking and key sleep features. To test for causal relationships, we then used behavioral, chemogenetic, or optogenetic methods to selectively increase rapid eye movement sleep (REMS) and measured behavioral and electrophysiological outcomes to probe for cellular and circuit mechanisms underlying REMS-mediated regulation of cocaine seeking. RESULTS: A selective set of REMS features was preferentially associated with the intensity of cue-induced cocaine seeking after drug withdrawal. Moreover, selectively increasing REMS time and continuity by environmental warming attenuated a withdrawal time-dependent intensification of cocaine seeking, or incubation of cocaine craving, suggesting that REMS may benefit withdrawal. Warming increased the activity of lateral hypothalamic melanin-concentrating hormone (MCH) neurons selectively during prolonged REMS episodes and counteracted cocaine-induced synaptic accumulation of calcium-permeable AMPA receptors in the nucleus accumbens-a critical substrate for incubation. Finally, the warming effects were partly mimicked by chemogenetic or optogenetic stimulations of MCH neurons during sleep, or intra-accumbens infusions of MCH peptide during the rat's inactive phase. CONCLUSIONS: REMS may encode individual vulnerability to relapse, and MCH neuron activities can be selectively targeted during REMS to reduce drug relapse.

Prefrontal inhibition drives formation and dynamic expression of probabilistic Pavlovian fear conditioning.

The association between cause and effect is usually probabilistic. Memories triggered by ambiguous cues may be altered or biased into a more negative perception in psychiatric diseases. Understanding the formation and modulation of this probabilistic association is important for revealing the nature of aversive memory and alterations in brain diseases. We found that 50% conditioned and unconditioned stimuli (CS-US) association during Pavlovian fear conditioning results in reduced fear responses and neural spiking in the dorsomedial prefrontal cortex (dmPFC) due to enhanced inhibition from dmPFC parvalbumin (PV) neurons. Formation of probabilistic memory is associated with increased synaptic inputs to PV-neurons and requires activation of ventral hippocampus, which detects CS-US mismatch during conditioning. Stress prior to conditioning impairs the formation of probabilistic memory by abolishing PV-neuronal plasticity, while stress prior to memory retrieval reverts enhanced PV-neuron activity. In conclusion, PV-neurons tailor learned responses to fit brain state at the moment of retrieval.

TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.

UAF1 deubiquitinase complexes facilitate NLRP3 inflammasome activation by promoting NLRP3 expression.

NOD-like receptor protein 3 (NLRP3) detects microbial infections or endogenous danger signals and activates the NLRP3 inflammasome, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases, and thereby needs to be tightly controlled. Deubiquitination of NLRP3 is considered a key step in NLRP3 inflammasome activation. However, the mechanisms by which deubiquitination controls NLRP3 inflammasome activation are unclear. Here, we show that the UAF1/USP1 deubiquitinase complex selectively removes K48-linked polyubiquitination of NLRP3 and suppresses its ubiquitination-mediated degradation, enhancing cellular NLRP3 levels, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. In addition, the UAF1/USP12 and UAF1/USP46 complexes promote NF-κB activation, enhance the transcription of NLRP3 and proinflammatory cytokines (including pro-IL-1ß, TNF, and IL-6) by inhibiting ubiquitination-mediated degradation of p65. Consequently, Uaf1 deficiency attenuates NLRP3 inflammasome activation and IL-1ß secretion both in vitro and in vivo. Our study reveals that the UAF1 deubiquitinase complexes enhance NLRP3 and pro-IL-1ß expression by targeting NLRP3 and p65 and licensing NLRP3 inflammasome activation.

The impact of D-cycloserine and sarcosine on in vivo frontal neural activity in a schizophrenia-like model.

BACKGROUND: N-methyl-D-aspartate receptor (NMDAR) hypofunction has been proposed to underlie the pathogenesis of schizophrenia. Specifically, reduced function of NMDARs leads to altered balance between excitation and inhibition which further drives neural network malfunctions. Clinical studies suggested that NMDAR modulators (glycine, D-serine, D-cycloserine and glycine transporter inhibitors) may be beneficial in treating schizophrenia patients. Preclinical evidence also suggested that these NMDAR modulators may enhance synaptic NMDAR function and synaptic plasticity in brain slices. However, an important issue that has not been addressed is whether these NMDAR modulators modulate neural activity/spiking in vivo. METHODS: By using in vivo calcium imaging and single unit recording, we tested the effect of D-cycloserine, sarcosine (glycine transporter 1 inhibitor) and glycine, on schizophrenia-like model mice. RESULTS: In vivo neural activity is significantly higher in the schizophrenia-like model mice, compared to control mice. D-cycloserine and sarcosine showed no significant effect on neural activity in the schizophrenia-like model mice. Glycine induced a large reduction in movement in home cage and reduced in vivo brain activity in control mice which prevented further analysis of its effect in schizophrenia-like model mice. CONCLUSIONS: We conclude that there is no significant impact of the tested NMDAR modulators on neural spiking in the schizophrenia-like model mice.

Elevated dopamine signaling from ventral tegmental area to prefrontal cortical parvalbumin neurons drives conditioned inhibition.

Conditioned inhibition is an important process to suppress learned responses for optimal adaptation, but its underlying biological mechanism is poorly understood. Here we used safety learning (SL)/fear discrimination after fear conditioning as a conditioned inhibition model because it demonstrates the essential properties of summation and retardation. Activity of the dorsomedial prefrontal cortex (dmPFC) parvalbumin (PV) neurons bidirectionally regulates spiking levels of dmPFC excitatory neurons and fear states. Responses to safety cues are increased in dopaminergic (DA) neurons in the ventral tegmental area (VTA) and in PV neurons in dmPFC after SL. Plasticity in the VTA is implicated, since SL requires activation of N-methyl-d-aspartate receptors. Furthermore, in a posttraumatic stress disorder model, impaired SL is associated with impaired potentiation of VTA DA neuron activity. Our results demonstrate a DA-dependent learning process that targets prefrontal inhibitory neurons for suppression of learned responses, and have implications for the pathogenesis and treatment of various psychiatric diseases.

Coding of "Home Cage" by PFC Neurons.

Contexts play critical roles in many important aspects of an animal's routine functions, such as the interpretation of incoming signals and retrieved memories. The roles played by prefrontal cortex (PFC) neurons in the coding of contexts have been largely studied in relation to aversive stimuli (such as foot shock in conditioned fear). Whether PFC neurons may code contexts that mice encounter in everyday life, such as their home cage, is poorly understood. Here, we report the identification of a subpopulation of ventral medial PFC (vmPFC) neurons which change their spike rates when mice enter or leave their home cages. Both increase (ON units) and decrease (OFF units) in spike rate were observed, with about 2/3 of neurons showing decrease and 1/3 showing increase. These changes were evident whenever transitions occur from home cage to a different environment regardless of the novelty of the environments. In addition, changes in firing rate were not affected when mice entering a context where fear conditioning had taken place after contextual or auditory/cued fear conditioning. Furthermore, we found that the differential spike rates of ON and OFF units appear to allow mice to recognize that they are inside their home cages. Together, vmPFC neural spiking appears to enable the encoding of "home cage".