Here, we present a synthetic biology approach to assess the social behavioral consequences of altered function of the Krüppel-associated box zinc finger protein (KZFP) interacting protein TRIM28 within the prefrontal cortex (PFC) of male and female mice. We reprogrammed natural TRIM28 WT by replacing the transcriptionally repressive domain with an enhanced transcriptional activation domain VP64-p65-Rta (TRIM28 VPR ), or by excising the transcriptional regulatory domain (TRIM28 NFD ). In vitro validation confirmed that TRIM28 WT represses, and TRIM28 VPR activates, the expression of a KZFP-regulated luciferase reporter gene. Upon intra-PFC viral-mediated delivery of TRIM28 variants, we observed that inversion of TRIM28 transcriptional control via HSV-TRIM28 VPR reduced the salience of novel social interaction for male and female mice while not affecting non-social behaviors. RNA-sequencing revealed HSV-TRIM28 VPR promoted transcriptional escape of all classes of TEs, particularly those located within intronic and distal enhancer regions of downregulated immune genes. HSV-TRIM28 VPR -driven social deficits were reversible by intra-PFC repletion of interferon cytokines. These novel data point to PFC KZFP-TRIM28 interactions as necessary to stabilize TEs to enable cis-regulation of key immune gene expression and enhance organismal capacity for complex, pro-social behaviors.

The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in rodent prefrontal cortex was previously demonstrated to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189VPR, which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT. Following viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, inversion of ZFP189-mediated transcription in this brain area, achieved by viral delivery of synthetic ZFP189VPR, precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. RNA sequencing of virally manipulated prefrontal cortex tissues reveals that ZFP189 transcription factors of opposing regulatory function (ZFP189WT versus ZFP189VPR) have opposite influence on the expression of genetic transposable elements as well as genes that participate in adaptive immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for complex social behaviors while regulating transposable element-rich regions of DNA and the expression of immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal’s capacity for functioning in social groups.

A more complete understanding of the molecular mechanisms by which substance use is encoded in the brain could illuminate novel strategies to treat substance use disorders, including cocaine use disorder (CUD). We have previously discovered that Zfp189, which encodes a Kruppel-associated box zinc finger protein (KZFP) transcription factor (TF), differentially accumulates in nucleus accumbens (NAc) Drd1+ and Drd2+ medium spiny neurons (MSNs) over the course of cocaine exposure and is causal in producing MSN functional and behavioral changes to cocaine(1). Here, we aimed to illuminate the brain cell-type specific molecular mechanisms through which this KZFP TF produces CUD-related brain changes, with emphasis on investigating transposable elements (TEs), since KZFPs like ZFP189 are known regulators of TEs(2-6). First, we annotated TEs in existing single nuclei RNA-sequencing (snRNAseq) datasets of rodents that were exposed to either acute or repeated cocaine. We discovered that expression of NAc TEs was dramatically altered by cocaine experience, the most sensitive NAc cell-type was MSNs, and TEs in Drd1+ MSNs were considerably more dynamic over the course of cocaine exposure than TEs in Drd2+ MSNs. To determine the causality of this TE dysregulation within NAc MSNs in cocaine-induced brain changes, we virally delivered conditional synthetic ZFP189 TFs of our own design to Drd1+ or Drd2+ MSNs. These synthetic ZFP189 TFs are capable of directly activating (ZFP189VPR) or repressing (ZFP189WT) brain TEs2. We discover that behavioral and cell morphological adaptations to cocaine are produced by activating TEs with ZFP189VPR in Drd1+ MSNs or stabilizing TEs with ZFP189WT in Drd2+ MSNs, revealing a persistent opponent process balanced across MSN subtypes and weighted by TE stability and consequent gene expression within MSN subtype. We next performed snRNAseq of the whole NAc virally manipulated with ZFP189 TFs. We observed that, relative to ZFP189WT, NAc manipulated with ZFP189VPR impeded cocaine-induced gene expression in NAc cell-types, including both Drd1+ and Drd2+ MSNs. Within either MSN subtype, the consequence of normal ZFP189 function was to enhance immune-related gene expression, and ZFP189VPR impeded these gene expression profiles. We finally performed cocaine intravenous self-administration to determine the consequence of NAc ZFP189-mediated transcriptional control on cocaine use behaviors. We observed that ZFP189VPR impeded any increases in active lever responses following a period forced cocaine abstinence. This research demonstrates that KZFP-mediated transcriptional repression of TEs within NAc MSNs is a causal molecular step in enabling gene expression and subsequent cellular and behavioral responses to cocaine use, and the use of ZFP189VPR in this work demonstrates cell-type specific mechanistic strategies to block CUD-related brain adaptations, which may inform future CUD treatments. ### Competing Interest Statement The authors have declared no competing interest.

The development of drug addiction is characterized by molecular changes in brain reward regions that lead to the transition from recreational to compulsive drug use. These neurobiological processes in brain reward regions, such as the nucleus accumbens (NAc), are orchestrated in large part by transcriptional regulation. Our group recently identified the transcription factor E2F3a as a novel regulator of cocaine's rewarding effects and gene expression regulation in the NAc of male mice. Despite this progress, no information is available about the role of E2F3a in regulating cocaine reward at the sex- and cell-specific levels. Here, we used male and female mice expressing Cre-recombinase in either D1- or D2-type medium spiny neurons (MSNs) combined with viral-mediated gene transfer to bidirectionally control levels of E2F3a in a cell-type-specific manner in the NAc during conditioned place preference (CPP) to cocaine. Our findings show that selective overexpression of E2F3a in D1-MSNs increased cocaine CPP in both male and female mice, whereas opposite effects were observed under knockdown conditions. In contrast, equivalent E2F3a manipulations in D2-MSNs had no significant effects. To further explore the role of E2F3a in sophisticated operant and motivated behaviors, we performed viral manipulations of all NAc neurons in combination with cocaine self-administration and behavioral economics procedures in rats and demonstrated that E2F3a regulates sensitivity aspects of cocaine seeking and taking. These results confirm E2F3a as a central substrate of cocaine reward and demonstrate that this effect is mediated in D1-MSNs, thereby providing increased knowledge of cocaine action at the transcriptional level.

Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with ∼3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.

Based on “reducing amyloid plaques in the brain”, the U.S. Food and Drug Administration has granted accelerated and full approval for two monoclonal anti-Alzheimer’s antibodies, aducanumab and lecanemab, respectively. Approval of a third antibody, donanemab, is pending. Moreover, lecanemab and donanemab are claimed to cause delay in the cognitive decline that characterizes the disease. We believe that these findings are subject to misinterpretation and statistical bias. Donanemab is claimed to cause removal of up to 86% of cerebral amyloid and 36% delay in cognitive decline compared to placebo. In reality, these are very small changes on an absolute scale and arguably less than what can be achieved with cholinesterase inhibitor/memantine therapy. Moreover, the "removal" of amyloid, based on the reduced accumulation of amyloid-PET tracer, most likely also reflects therapy-related tissue damage. This would also correlate with the minimal clinical effect, the increased frequency of amyloid-related imaging abnormalities, and the accelerated loss of brain volume in treated compared to placebo patients observed with these antibodies. We recommend halting approvals of anti-AD antibodies until these issues are fully understood to ensure that antibody treatment does not cause more harm than benefit to patients.

Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrated that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8–10-week-old male C57BL/6 J mice were randomly assigned to groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing, respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced all stressed and single housed mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or single housed mice. In sum, although we confirm elevated vHipp protein levels of H1x associate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are associated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.

The amyloid cascade hypothesis for Alzheimer’s disease is still alive, although heavily challenged. Effective anti-amyloid immunotherapy would confirm the hypothesis’ claim that the protein amyloid-beta is the cause of the disease. Two antibodies, aducanumab and lecanemab, have been approved by the U.S. Food and Drug Administration, while a third, donanemab, is under review. The main argument for the FDA approvals is a presumed therapy-induced removal of cerebral amyloid deposits. Lecanemab and donanemab are also thought to cause some statistical delay in the determination of cognitive decline. However, clinical efficacy that is less than with conventional treatment, selection of amyloid-positive trial patients with non-specific amyloid-PET imaging, and uncertain therapy-induced removal of cerebral amyloids in clinical trials cast doubt on this anti-Alzheimer’s antibody therapy and hence on the amyloid hypothesis, calling for a more thorough investigation of the negative impact of this type of therapy on the brain.

Distinguishing the brain mechanisms affected by distinct addictive drugs may inform targeted therapies against specific substance use disorders (SUDs). Here, we explore the function of a drug-associated, transcriptionally repressive transcription factor (TF), ZFP189, whose expression in the nucleus accumbens (NAc) facilitates cocaine-induced molecular and behavioral adaptations. To uncover the necessity of ZFP189-mediated transcriptional control in driving cocaine-induced behaviors, we created synthetic ZFP189 TFs of distinct transcriptional function, including ZFP189VPR, which activates the expression of target genes and exerts opposite transcriptional control to the endogenously repressive ZFP189. By virally delivering synthetic ZFP189 TFs to the NAc of mice, we discover that the transcriptional control exerted by synthetic or endogenous ZFP189 solely alters behavioral adaptations to cocaine but not morphine, saline, or sucrose. Further, these synthetic ZFP189 TFs are only capable of producing gene-expression changes in rodents exposed to cocaine, but not morphine or saline. In these cocaine exposed mice, the gene-expression profile produced by ZFP189VPR is inversely related to the cocaine-induced transcriptional response, as characterized by Upstream Regulator Analysis in Ingenuity Pathway Analysis. Lastly, we demonstrate that NAc ZFP189WT increases vulnerability to cocaine reinforcement through selective sensitization to the reinforcing effects of small cocaine doses. In contrast, ZFP189VPR treated mice do not experience changes in cocaine sensitivity and had lower rates of cocaine self-administration. Collectively, this research describes the brain mechanisms by which a TF specifically coordinates the molecular adaptations that produce increased cocaine addiction-like behaviors. The use of synthetic ZFP189VPR uncovers novel strategies for therapeutic interventions to potentially halt these cocaine-induced transcriptional processes.

ABSTRACTThe complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here we implicate in this process a novel nuclear receptor transcription factor, Retinoid X Receptor Alpha (RXRα), which we identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that, while its own expression remains unaltered after cocaine exposure, RXRα controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXRα regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXRα in promoting drug addiction, and paves the way for future studies of rexinoid signaling in psychiatric disease states.

The recently announced revision of the Alzheimer's disease (AD) diagnostic ATN classification adds to an already existing disregard for clinical assessment the rejection of image -based in vivo assessment of the brain's condition. The revision suggests that the diagnosis of AD should be based solely on the presence of cerebral amyloid-beta and tau, indicated by the "A" and "T". The "N", which stands for neurodegeneration - detected by imaging - should no longer be given importance, except that A+ +/- T + = AD with amyloid PET being the main method for demonstrating A+ . We believe this is an artificial and misleading suggestion. It is artificial because it relies on biomarkers whose significance remains obscure and where the detection of "A" is based on a never-validated PET method using a tracer that marks much more than amyloid-beta. It is misleading because many patients without dementia will be falsely classified as having AD, but nonetheless candidates for passive immunotherapy, which may be more harmful than beneficial, and sometimes fatal.

After the CLARITY-AD clinical trial results of lecanemab were interpreted as positive, and supporting the amyloid hypothesis, the drug received accelerated Food and Drug Administration approval. However, we argue that benefits of lecanemab treatment are uncertain and may yield net harm for some patients, and that the data do not support the amyloid hypothesis. We note potential biases from inclusion, unblinding, dropouts, and other issues. Given substantial adverse effects and subgroup heterogeneity, we conclude that lecanemab's efficacy is not clinically meaningful, consistent with numerous analyses suggesting that amyloid-β and its derivatives are not the main causative agents of Alzheimer's disease dementia.

BACKGROUND:Major depressive disorder is one of the most commonly diagnosed mental illnesses worldwide, with a higher prevalence in women than in men. Although currently available pharmacological therapeutics help many individuals, they are not effective for most. Animal models have been important for the discovery of molecular alterations in stress and depression, but difficulties in adapting animal models of depression for females has impeded progress in developing novel therapeutic treatments that may be more efficacious for women.METHODS:Using the California mouse social defeat model, we took a multidisciplinary approach to identify stress-sensitive molecular targets that have translational relevance for women. We determined the impact of stress on transcriptional profiles in male and female California mouse nucleus accumbens (NAc) and compared these results with data from postmortem samples of the NAc from men and women diagnosed with major depressive disorder.RESULTS:Our cross-species computational analyses identified Rgs2 (regulator of G protein signaling 2) as a transcript downregulated by social defeat stress in female California mice and in women with major depressive disorder. RGS2 plays a key role in signal regulation of neuropeptide and neurotransmitter receptors. Viral vector-mediated overexpression of Rgs2 in the NAc restored social approach and sucrose preference in stressed female California mice.CONCLUSIONS:These studies show that Rgs2 acting in the NAc has functional properties that translate to changes in anxiety- and depression-related behavior. Future studies should investigate whether targeting Rgs2 represents a novel target for treatment-resistant depression in women.

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

Relapse remains a major challenge to the treatment of cocaine addiction. Recent studies suggested that the trace amine-associated receptor 1 (TAAR1) could be a promising target to treat cocaine addiction and relapse; however, the underlying mechanism remains unclear. Here, we aimed to investigate the neural mechanism underlying the role of TAAR1 in the drug priming-induced reinstatement of cocaine-seeking behavior in rats, an animal model of cocaine relapse. We focused on the shell subregion of nucleus accumbens (NAc), a key brain region of the brain reward system. We found that activation of TAAR1 by systemic and intra-NAc shell administration of the selective TAAR1 agonist RO5166017 attenuated drug-induced reinstatement of cocaine-seeking and prevented drug priming-induced CaMKIIα activity in the NAc shell. Activation of TAAR1 dampened the CaMKIIα/GluR1 signaling pathway in the NAc shell and reduced AMPAR-EPSCs on the NAc slice. Microinjection of the selective TAAR1 antagonist EPPTB into the NAc shell enhanced drug-induced reinstatement as well as potentiated CaMKIIα activity in the NAc shell. Furthermore, viral-mediated expression of CaMKIIα in the NAc shell prevented the behavioral effects of TAAR1 activation. Taken together, our findings indicate that TAAR1 regulates drug-induced reinstatement of cocaine-seeking by negatively regulating CaMKIIα activity in the NAc. Our findings elucidate a novel mechanism of TAAR1 in regulating drug-induced reinstatement of cocaine-seeking and further suggests that TAAR1 is a promising target for the treatment of cocaine relapse.

BACKGROUND: Over the course of chronic drug use, brain transcriptional neuroadaptation is thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB (cAMP response element binding protein) within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuro-adaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within the NAc of mice.METHODS: To investigate the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 constructs capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice.RESULTS: We observed that CREB binding to the Zfp189 promoter increased Zfp189 expression and diminished the reinforcing responses to cocaine. Furthermore, we showed that NAc Zfp189 expression increased within D1 medium spiny neurons in response to acute cocaine but increased in both D1-and D2-expressing medium spiny neurons in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiated electrophysiological activity of D1-and D2-expressing medium spiny neurons, recapitulating the known effect of CREB on these neurons. Finally, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors.CONCLUSIONS: Together, these findings point to the CREB-Zfp189 interaction within the NAc Drd21 neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.

With an incidence of -1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.

Drugs of abuse regulate the activity of the mesolimbic dopamine (DA) system, and drug-induced changes in ventral tegmental area (VTA) cellular activity and gene regulation are linked to behavioral outputs associated with addiction. Previous work from our lab determined that VTA serum- and glucocorticoid-inducible kinase 1 (SGK1) transcription and catalytic activity were increased by repeated cocaine administration; however, it was unknown if these biochemical changes contributed to cocaine-elicited behaviors. Using transgenic and viral-mediated manipulations, we investigated the role of VTA SGK1 catalytic activity in regulating cocaine conditioned place preference and self-administration. We showed intra-VTA infusion of a catalytically inactive SGK1 mutant (K127Q) significantly decreased cocaine conditioned place preference (CPP). Further, we found that K127Q expression in VTA DA neurons significantly decreased cocaine CPP, while this same manipulation in VTA GABA neurons had no effect. However, blunted VTA DA SGK1 catalytic activity did not alter cocaine self-administration. Altogether, these studies identify the specific VTA cells critical for SGK1-mediated effects on cocaine CPP but not self-administration.

BACKGROUND: The onset and persistence of addiction phenotypes are, in part, mediated by transcriptional mechanisms in the brain that affect gene expression and, subsequently, neural circuitry. Delta FosB is a transcription factor that accumulates in the nucleus accumbens (NAc)-a brain region responsible for coordinating reward and motivation-after exposure to virtually every known rewarding substance, including cocaine and opioids. Delta FosB has also been shown to directly control gene transcription and behavior downstream of both cocaine and opioid exposure, but with potentially different roles in D1 and D2 medium spiny neurons (MSNs) in NAc. METHODS: To clarify MSN subtype-specific roles for Delta FosB and investigate how these coordinate the actions of distinct classes of addictive drugs in NAc, we developed a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-based epigenome editing tool to induce endogenous Delta FosB expression in vivo in the absence of drug exposure. After inducing Delta FosB in D1-or D2-MSNs or both, we performed RNA sequencing on bulk male and female NAc tissue (n = 6-8/group). RESULTS: We found that Delta FosB induction elicits distinct transcriptional profiles in NAc by MSN subtype and by sex, establishing for the first time that AFosB mediates different transcriptional effects in males versus females. We also demonstrated that changes in D1-MSNs, but not those in D2-MSNs or both, significantly recapitulate changes in gene expression induced by cocaine self-administration. CONCLUSIONS: Together, these findings demonstrate the efficacy of a novel molecular tool for studying cell type- specific transcriptional mechanisms and shed new light on the activity of Delta FosB, a critical transcriptional regulator of drug addiction.