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Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections., Programmed death-ligand 1 (PD-L1) is involved in the inhibition of antigen specific T cells via ligation of programmed death 1 (PD-1). Here, the authors show checkpoint inhibition by use of small molecule inhibition of PD-L1 which in a humanised mouse model was shown to restore T cell responses and reduced tumour burden.

Long-term memory formation requires coordinated regulation of gene expression and persistent changes in cell function. For decades, research has implicated histone modifications in regulating chromatin compaction necessary for experience-dependent changes to gene expression and cell function during memory formation. Recent evidence suggests that another epigenetic mechanism, ATP-dependent chromatin remodeling, works in concert with the histone-modifying enzymes to produce large-scale changes to chromatin structure. This review examines how histone-modifying enzymes and chromatin remodelers restructure chromatin to facilitate memory formation. We highlight the emerging evidence implicating ATP-dependent chromatin remodeling as an essential mechanism that mediates activity-dependent gene expression, plasticity, and cell function in developing and adult brains. Finally, we discuss how studies that target chromatin remodelers have expanded our understanding of the role that these complexes play in substance use disorders.

Epigenetic mechanisms result in persistent changes at the cellular level that can lead to long-lasting behavioral adaptations. Nucleosome remodeling is a major epigenetic mechanism that has not been well explored with regards to drug-seeking behaviors. Nucleosome remodeling is performed by multi-subunit complexes that interact with DNA or chromatin structure and possess an ATP-dependent enzyme to disrupt nucleosome-DNA contacts and ultimately regulate gene expression. Calcium responsive transactivator (CREST) is a transcriptional activator that interacts with enzymes involved in both histone acetylation and nucleosome remodeling. Here, we examined the effects of knocking down CREST in the nucleus accumbens (NAc) core on drug-seeking behavior and synaptic plasticity in male mice as well as drug-seeking in male rats. Knocking down CREST in the NAc core results in impaired cocaine-induced conditioned place preference (CPP) as well as theta-induced long-term potentiation in the NAc core. Further, similar to the CPP findings, using a self-administration procedure, we found that CREST knockdown in the NAc core of male rats had no effect on instrumental responding for cocaine itself on a first-order schedule, but did significantly attenuate responding on a second-order chain schedule, in which responding has a weaker association with cocaine. Together, these results suggest that CREST in the NAc core is required for cocaine-induced CPP, synaptic plasticity, as well as cocaine-seeking behavior.SIGNIFICANCE STATEMENTThis study demonstrates a key role for the role of Calcium responsive transactivator (CREST), a transcriptional activator, in the nucleus accumbens (NAc) core with regard to cocaine-induced conditioned place preference (CPP), self-administration (SA), and synaptic plasticity. CREST is a unique transcriptional regulator that can recruit enzymes from two different major epigenetic mechanisms: histone acetylation and nucleosome remodeling. In this study we also found that the level of potentiation in the NAc core correlated with whether or not animals formed a CPP. Together the results indicate that CREST is a key downstream regulator of cocaine action in the NAc.

Propensity to relapse, even following long periods of abstinence, is a key feature in substance use disorders. Relapse and relapse-like behaviors are known to be induced, in part, by re-exposure to drug-associated cues. Yet, while many critical nodes in the neural circuitry contributing to relapse have been identified and studied, a full description of the networks driving reinstatement of drug-seeking behaviors is lacking. One area that may provide further insight to the mechanisms of relapse is the habenula complex, an epithalamic region composed of lateral and medial (MHb) substructures, each with unique cell and target populations. Although well conserved across vertebrate species, the functions of the MHb are not well understood. Recent research has demonstrated that the MHb regulates nicotine aversion and withdrawal. However, it remains undetermined whether MHb function is limited to nicotine and aversive stimuli or if MHb circuit regulates responses to other drugs of abuse. Advances in circuit-level manipulations now allow for cell-type and temporally specific manipulations during behavior, specifically in spatially restrictive brain regions, such as the MHb. In this study, we focus on the response of the MHb to reinstatement of cocaine-associated behavior, demonstrating that cocaine-primed reinstatement of conditioned place preference engages habenula circuitry. Using chemogenetics, we demonstrate that MHb activity is sufficient to induce reinstatement behavior. Together, these data identify the MHb as a key hub in the circuitry underlying reinstatement and may serve as a target for regulating relapse-like behaviors.

Our memory for time is a fundamental ability that we use to judge the duration of events, put our experiences into a temporal context, and decide when to initiate actions. The medial entorhinal cortex (MEC), with its direct projections to the hippocampus, has been proposed to be the key source of temporal information for hippocampal time cells. However, the behavioral relevance of such temporal firing patterns remains unclear, as most of the paradigms used for the study of temporal processing and time cells are either spatial tasks or tasks for which MEC function is not required. In this study, we asked whether the MEC is necessary for rats to perform a time duration discrimination task (TDD), in which rats were trained to discriminate between 10-s and 20-s delay intervals. After reaching a 90% performance criterion, the rats were assigned to receive an excitotoxic MEC-lesion or sham-lesion surgery. We found that after recovering from surgery, rats with MEC lesions were impaired on the TDD task in comparison to rats with sham lesions, failing to return to criterion performance. Their impairment, however, was specific to the longer, 20-s delay trials. These results indicate that time processing is dependent on MEC neural computations only for delays that exceed 10 s, perhaps because long-term memory resources are needed to keep track of longer time intervals.

Cancer cachexia is a highly prevalent condition associated with poor quality of life and reduced survival1. Tumor-induced perturbations in the endocrine, immune and nervous systems drive anorexia and catabolic changes in adipose tissue and skeletal muscle, hallmarks of cancer cachexia2-4. However, the molecular mechanisms driving cachexia remain poorly defined, and there are currently no approved drugs for the condition. Elevation in circulating growth differentiation factor 15 (GDF15) correlates with cachexia and reduced survival in patients with cancer5-8, and a GDNF family receptor alpha like (GFRAL)-Ret proto-oncogene (RET) signaling complex in brainstem neurons that mediates GDF15-induced weight loss in mice has recently been described9-12. Here we report a therapeutic antagonistic monoclonal antibody, 3P10, that targets GFRAL and inhibits RET signaling by preventing the GDF15-driven interaction of RET with GFRAL on the cell surface. Treatment with 3P10 reverses excessive lipid oxidation in tumor-bearing mice and prevents cancer cachexia, even under calorie-restricted conditions. Mechanistically, activation of the GFRAL-RET pathway induces expression of genes involved in lipid metabolism in adipose tissues, and both peripheral chemical sympathectomy and loss of adipose triglyceride lipase protect mice from GDF15-induced weight loss. These data uncover a peripheral sympathetic axis by which GDF15 elicits a lipolytic response in adipose tissue independently of anorexia, leading to reduced adipose and muscle mass and function in tumor-bearing mice.

A key component of combating substance use disorders is understanding the neural mechanisms that support drug reward. Tasks such as self-administration assess the reinforcing properties of a drug using a learned behavior but require numerous training sessions and surgery. In comparison, the conditioned place preference (CPP) task assesses reward with little training, without costly surgeries, and confounds that accompany the use of anesthesia or pain-relieving drugs. The CPP task contains three phases: pretest, conditioning, and posttest. During the pretest, mice are allowed to explore a three-compartment apparatus. The two outer compartments contain unique olfactory, tactile, and visual cues whereas the middle compartment is used as an entrance and exit for the mice on test days. During conditioning, mice receive cocaine before being confined to one of the outer compartments. The following day, mice are given saline then confined to the other outer compartment. These pairings are then repeated once. At posttest, mice are permitted to freely explore all compartments in a drug-free state while the time spent in each compartment is recorded. A CPP score is calculated for both the pretest and posttest by comparing the time spent in the cocaine-paired and saline-paired compartments. Enhancements in the CPP score from the pretest to the posttest serve as a measure of the rewarding property of the cocaine. This task offers several notable advantages: 1) the simultaneous recording of locomotor activity and reward, which may utilize different neural mechanisms, 2) the three-compartment CPP setup removes the bias that can be observed in a two-compartment design, and 3) use of multimodal cues support the acquisition of a robust preference in a variety of mouse strains.

Propensity to relapse following long periods of abstinence is a key feature of substance use disorder. Drugs of abuse, such as cocaine, cause long-term changes in the neural circuitry regulating reward, motivation, and memory processes through dysregulation of various molecular mechanisms, including epigenetic regulation of activity-dependent gene expression. Underlying drug-induced changes to neural circuit function are the molecular mechanisms regulating activity-dependent gene expression. Of note, histone acetyltransferases and histone deacetylases (HDACs), powerful epigenetic regulators of gene expression, are dysregulated following both acute and chronic cocaine exposure and are linked to cocaine-induced changes in neural circuit function. To better understand the effect of drug-induced changes on epigenetic function and behavior, we investigated HDAC3-mediated regulation of Nr4a2/Nurr1 in the medial habenula, an understudied pathway in cocaine-associated behaviors. Nr4a2, a transcription factor critical in cocaine-associated behaviors and necessary for MHb development, is enriched in the cholinergic cell-population of the MHb; yet, the role of NR4A2 within the MHb in the adult brain remains elusive. Here, we evaluated whether epigenetic regulation of Nr4a2 in the MHb has a role in reinstatement of cocaine-associated behaviors. We found that HDAC3 disengages from Nr4a2 in the MHb in response to cocaine-primed reinstatement. Whereas enhancing HDAC3 function in the MHb had no effect on reinstatement, we found, using a dominant-negative splice variant (NURR2C), that loss of NR4A2 function in the MHb blocked reinstatement behaviors. These results show for the first time that regulation of NR4A2 function in the MHb is critical in relapse-like behaviors.

Histone acetylation is a fundamental epigenetic mechanism that is dynamically regulated during memory formation. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) compete to modulate histone acetylation, allowing for rapid changes in acetylation in response to a learning event. HDACs are known to be powerful negative regulators of memory formation, but it is not clear whether this function depends on HDAC enzymatic activity per se. Here, we tested whether the enzymatic activity of an individual Class I HDAC, HDAC3, has a role in fear memory formation in subregions of the hippocampus and amygdala. We found that fear conditioning drove expression of the immediate early genes cFos and Nr4a2 in the hippocampus, which coincided with reduced HDAC3 occupancy at these promoters. Using a dominant-negative, deacetylase-dead point mutant virus (AAV-HDAC3(Y298H)-v5), we found that selectively blocking HDAC3 deacetylase activity in either the dorsal hippocampus or basal nucleus of the amygdala enhanced context fear without affecting tone fear. Blocking HDAC3 activity in the lateral nucleus of the amygdala, on the other hand, enhanced tone, but not context fear memory. These results show for the first time that the enzymatic activity of HDAC3 functions to negatively regulate fear memory formation. Further, HDAC3 activity regulates different aspects of fear memory in the basal and lateral subregions of the amygdala. Thus, the deacetylase activity of HDAC3 is a powerful negative regulator of fear memory formation in multiple subregions of the fear circuit.

Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. We demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains.

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and learning-induced expression of the circadian genePer1in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampalPer1is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation.

Histone deacetylases (HDACs) are chromatin modifying enzymes that have been implicated as powerful negative regulators of memory processes. HDAC3 has been shown to play a pivotal role in long-term memory for object location as well as the extinction of cocaine-associated memory, but it is unclear whether this function depends on the deacetylase domain of HDAC3. Here, we tested whether the deacetylase domain of HDAC3 has a role in object location memory formation as well as the formation and extinction of cocaine-associated memories. Using a deacetylase-dead point mutant of HDAC3, we found that selectively blocking HDAC3 deacetylase activity in the dorsal hippocampus enhanced long-term memory for object location, but had no effect on the formation of cocaine-associated memory. When this same point mutant virus of HDAC3 was infused into the prelimbic cortex, it failed to affect cocaine-associated memory formation. With regards to extinction, impairing the HDAC3 deacetylase domain in the infralimbic cortex had no effect on extinction, but a facilitated extinction effect was observed when the point mutant virus was delivered to the dorsal hippocampus. These results suggest that the deacetylase domain of HDAC3 plays a selective role in specific brain regions underlying long-term memory formation of object location as well as cocaine-associated memory formation and extinction.

Flipped instruction using online enrichment is a popular way to enhance active learning in the laboratory setting. Graduate student teaching assistants at University of California, Irvine flipped an upper division undergraduate neurobiology and behavior lab using the new online software platform “Rocketmix.” The following research study compares the impact of pre-lab online instruction (front flipping) and post-lab online instruction (back flipping) on student exam performance. We describe a novel method for unbiased categorization of exam questions by degree of difficulty. Multi-choice instruction encourages students to consider all distractors and discourages verbal cues and process of elimination techniques. Eighteen identical questions were evenly distributed across exam versions with multiple choice instruction (single answer) or a more challenging multi-choice instruction (more than one answer). Student performance on multiple choice questions were used to categorize the degree of difficulty of questions that were presented in multi-choice format. Our findings reveal that pre-lab instruction resulted in better student performance compared with post-lab instruction on questions of moderate difficulty. This effect was significant for both male and female students. Student survey data on the flipped lab format is provided, indicating that students appreciated the online instructional modules, finding them both informative and useful during lab exercises and exams.

It has been known for numerous decades that gene expression is required for long-lasting forms of memory. In the past decade, the study of epigenetic mechanisms in memory processes has revealed yet another layer of complexity in the regulation of gene expression. Epigenetic mechanisms do not only provide complexity in the protein regulatory complexes that control coordinate transcription for specific cell function, but the epigenome encodes critical information that integrates experience and cellular history for specific cell functions as well. Thus, epigenetic mechanisms provide a unique mechanism of gene expression regulation for memory processes. This may be why critical negative regulators of gene expression, such as histone deacetylases (HDACs), have powerful effects on the formation and persistence of memory. For example, HDAC inhibition has been shown to transform a subthreshold learning event into robust long-term memory and also generate a form of long-term memory that persists beyond the point at which normal long-term memory fails. A key question that is explored in this review, from a learning and memory perspective, is whether stress-dependent signaling drives the formation and persistence of long-term memory via HDAC-dependent mechanisms.

The present experiments examined the effects of prazosin, a selective α1-adrenergic receptor antagonist, on the development of methamphetamine conditioned hyperactivity and context-specific sensitization. Mice received an injection of vehicle (distilled water) or prazosin (0.5, 1.0 or 2.0 mg/kg) 30 minutes prior to a second injection of vehicle (saline) or methamphetamine (1.0 mg/kg) during the conditioning sessions (Experiment 1). Following the conditioning sessions, mice were tested for conditioned hyperactivity and then tested for context-specific sensitization. In subsequent experiments, mice received an injection of vehicle (distilled water) or prazosin (2.0 mg/kg) immediately (Experiment 2) or 24 hours (Experiment 3) after the conditioning sessions and then tested for conditioned hyperactivity and context-specific sensitization. Prazosin dose-dependently blocked the development of methamphetamine conditioned hyperactivity and context-specific sensitization when administered prior to the methamphetamine during the conditioning phase; however nonspecific motor impairments also were observed (Experiment 1). Immediate (Experiment 2), but not the 24-hour delay (Experiment 3), post-session administration of prazosin attenuated the development of methamphetamine conditioned hyperactivity and context-specific sensitization. Nonspecific motor impairments were not observed in these latter experiments. Collectively, these results suggest that the α1-adrenergic receptor mediates the development of methamphetamine-conditioned hyperactivity and context-specific sensitization, perhaps by altering memory consolidation and/or reconsolidation processes.

Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn–HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn–HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn–HM3D impaired LTP and hSyn–HM4D facilitated LTP. Follow-up experiments indicated that the hSyn–HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn–HM4D-mediated increase of fEPSP is induced by a reduction in GABAAreceptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα–HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα–HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα–HM3D hippocampal slices, whereas slices from CaMKIIα–HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP.SIGNIFICANCE STATEMENTThis study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally modulating the hippocampus in not only a hippocampus-dependent task but also in hippocampal synaptic plasticity. This is the first study to evaluate the effects of DREADD-mediated inhibition and excitation in hippocampal long-term potentiation. More specifically, this study evaluates the effect of promoter-specific expression of DREADD viruses in a heterogenic cell population, which revealed surprising effects of different promoters. With chemogenetics becoming a more ubiquitous tool throughout studies investigating circuit-specific function, these data are of broad interest to the neuroscientific community because we have shown that promoter-specific effects can drastically alter synaptic function within a specific region, without parallel changes at the level of behavior.