BDNF plays a crucial role in shaping the structure and function of neurons. In rodents, BDNF signaling in the dorsolateral striatum (DLS) is part of an endogenous pathway that protects against the development of phenotypes associated with alcohol use. Dysregulation of BDNF levels in the cortex or dysfunction of BDNF/TrkB signaling in the DLS of rodents results in the escalation of alcohol drinking and compulsive alcohol intake. The major source of BDNF in the striatum is the prefrontal cortex. We identified a small ensemble of BDNF-positive neurons in the mouse ventrolateral orbitofrontal cortex (vlOFC), a region implicated in alcohol use disorder (AUD), that extend axonal projections to the DLS, which is associated with alcohol drinking behaviors. We speculated that BDNF in vlOFC-to-DLS circuit may play a role in limiting alcohol drinking and that heavy alcohol intake disrupts this protective pathway. We found that BDNF expression is reduced in the vlOFC of male but not female mice after long-term cycles of binge alcohol drinking and withdrawal. We further discovered that overexpression of BDNF in vlOFC-to-DLS but not in vlOFC-to-dorsomedial striatum (DMS) or M2 motor cortex-to-DLS circuit reduces alcohol but not sucrose intake and preference. We further showed that BDNF in vlOFC-to DLS reduces alcohol self-administration, alcohol seeking, and relapse. Finally, we found that systemic administration of BDNF receptor TrkB agonist, LM22A-4, dampens habitual alcohol seeking. Together, our data suggest that BDNF in a small ensemble of vlOFC-to-DLS neurons may gate alcohol drinking behaviors by attenuating habitual alcohol seeking.

Prosapip1 is a brain-specific protein, encoded by Lzts3, localized to the postsynaptic density, where it promotes dendritic spine maturation in primary hippocampal neurons. However, nothing is known about the role of Prosapip1 in vivo. To examine this, we utilized the Cre-loxP system to develop a Prosapip1 neuronal knockout mouse. We found that Prosapip1 controls the synaptic localization of its binding partner SPAR, along with PSD-95 and the GluN2B subunit of the NMDA receptor (NMDAR) in the dorsal hippocampus (dHP). We next sought to identify the potential contribution of Prosapip1 to the activity and function of the NMDAR and found that Prosapip1 plays an important role in NMDAR-mediated transmission and long-term potentiation (LTP) in the CA1 region of the dHP. As LTP is the cellular hallmark of learning and memory, we examined the consequences of neuronal knockout of Prosapip1 on dHP-dependent memory. We found that global or dHP-specific neuronal knockout of Prosapip1 caused a deficit in learning and memor,y whereas developmental, locomotor, and anxiety phenotypes were normal. Taken together, Prosapip1 in the dHP promotes the proper localization of synaptic proteins which, in turn, facilitates LTP driving recognition, social, and spatial learning and memory.

mTORC1 promotes protein translation, learning and memory, and neuroadaptations that underlie alcohol use disorder (AUD). The mechanisms underlying alcohol-mediated mTORC1-dependent neuroadaptations that drive AUD are not well understood. We report that activation of mTORC1 in the nucleus accumbens (NAc) D1 neurons of male mice consuming alcohol results in paradoxical mTORC1-dependent repression of translation of transcripts, including Aldolase A, an essential enzyme in glycolysis. We further show that mTORC1-dependent Aldolase A translation repression in D1 neurons is mediated through upregulation of miR-34a-5p expression. Alcohol-mediated mTORC1 repression of Aldolase A translation in D1 neurons inhibits glycolysis in the NAc. Finally, we report that overexpression of miR-34a-5p in D1 NAc neurons increases, whereas systemic administration of L-lactate, the final product of glycolysis, attenuates excessive alcohol intake. Our data suggest that alcohol promotes paradoxical actions of mTORC1 on translation and glycolysis which in turn drive excessive alcohol use.

ABSTRACT The kinase mechanistic target of rapamycin complex 1 (mTORC1) plays an essential role in learning and memory by promoting mRNA to protein translation of a subset of synaptic proteins at dendrites. We generated a large body of data in male rodents indicating that mTORC1 is critically involved in mechanisms that promote numerous adverse behaviors associated with alcohol use disorder (AUD) including heavy alcohol use. For example, we found that mTORC1 is activated in the nucleus accumbens (NAc) and orbitofrontal cortex (OFC) of male mice and rats that were subjected to 7 weeks of intermittent access to 20% alcohol two-bottle choice (IA20%2BC). We further showed that systemic or intra-NAc administration of the selective mTORC1 inhibitor, rapamycin, decreases alcohol seeking and drinking, whereas intra-OFC administration of rapamycin reduces alcohol seeking and habit in male rats. This study aimed to assess mTORC1 activation in these corticostriatal regions of female mice and to determine whether the selective mTORC1 inhibitor, rapamycin, can be used to reduce heavy alcohol use in female mice. We found that mTORC1 is not activated by 7 weeks of intermittent 20% alcohol binge drinking and withdrawal in the NAc and OFC. Like in males, mTORC1 signaling was not activated by chronic alcohol intake and withdrawal in the medial prefrontal cortex (mPFC) of female mice. Interestingly, Pearson correlation comparisons revealed that the basal level of mTORC1 activation between the two prefrontal regions, OFC and mPFC were correlated and that the drinking profile predicts the level of mTORC1 activation in the mPFC after 4-hour binge drinking. Finally, we report that administration of rapamycin does not attenuate heavy alcohol drinking in female animals. Together, our results suggest a sex-dependent contribution of mTORC1 to the neuroadaptation that drives alcohol use and abuse.

The small G-protein Rac1 promotes the formation of filamentous actin (F-Actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice, or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an AAV expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning. Significance Statement:Addiction, including alcohol use disorder, is characterized by molecular and cellular adaptations that promote maladaptive behaviors. We found that Rac1 was activated by alcohol in the dorsomedial striatum (DMS) of male mice. We show that alcohol-mediated Rac1 signaling is responsible for alterations in actin dynamics and neuronal morphology. We also present data to suggest that Rac1 is important for alcohol-associated learning processes. These results suggest that Rac1 in the DMS is an important contributor to adaptations that promote alcohol use disorder.

Rett syndrome (RTT) is an X-linked neurogenetic disorder caused by mutations of the MECP2 (methyl-CpG-binding protein 2) gene. Over two decades of work established MeCP2 as a protein with pivotal roles in the regulation of the epigenome, neuronal physiology, synaptic maintenance, and behaviour. Given the genetic aetiology of RTT and the proof of concept of its reversal in a mouse model, considerable efforts have been made to design therapeutic approaches to re-express MeCP2. By being at the forefront of the development of innovative gene therapies, research on RTT is of paramount importance for the treatment of monogenic neurological diseases. Here we discuss the recent advances and challenges of promising genetic strategies for the treatment of RTT including gene replacement therapies, gene/RNA editing strategies, and reactivation of the silenced X chromosome.

AbstractmTORC1 promotes protein translation, learning and memory, and neuroadaptations that underlie alcohol use and abuse. We report that activation of mTORC1 in the nucleus accumbens (NAc) of mice consuming alcohol promotes the translation of microRNA (miR) machinery components and the upregulation of microRNAs (miRs) expression including miR-34a-5p. In parallel, we detected a paradoxical mTORC1-dependent repression of translation of transcripts including Aldolase A, an essential glycolytic enzyme. We found that miR-34a-5p in the NAc targets Aldolase A for translation repression and promotes alcohol intake. Our data further suggest that glycolysis is inhibited in the NAc manifesting in an mTORC1-dependent attenuation of L-lactate, the end product of glycolysis. Finally, we show that systemic administration of L-lactate attenuates mouse excessive alcohol intake. Our data suggest that alcohol promotes paradoxical actions of mTORC1 on translation and glycolysis which in turn drive excessive alcohol use.

AbstractBrain-derived neurotrophic factor (BDNF) is released from axon terminals originating in the cerebral cortex onto striatal neurons. Here, we characterized BDNF neurons in the corticostriatal circuitry. First, we usedBDNF-Cre and Ribotag transgenic mouse lines to label BDNF-positive neurons in the cortex and detectedBDNFexpression in all the subregions of the prefrontal cortex (PFC). Next, we used a retrograde viral tracing strategy, in combination withBDNF-Cre knock-in mice, to map the cortical outputs of BDNF neurons in the dorsomedial and dorsolateral striatum (DMS and DLS, respectively). We found thatBDNF-expressing neurons located in the medial prefrontal cortex (mPFC) project mainly to the DMS, and those located in the primary and secondary motor cortices (M1 and M2, respectively) and agranular insular cortex (AI) project mainly to the DLS. In contrast,BDNF-expressing orbitofrontal cortical (OFC) neurons differentially target the dorsal striatum (DS) depending on their mediolateral and rostrocaudal location. Specifically, the DMS is mainly innervated by the medial and ventral part of the orbitofrontal cortex (MO and VO, respectively), whereas the DLS receives projections specifically from the lateral part of the OFC (LO). Together, our study uncovers previously unknown BDNF corticostriatal circuitries. These findings could have important implications for the role of BDNF signaling in corticostriatal pathways.

Rett syndrome (RTT) is an X-linked neurogenetic disorder caused by mutations of the MECP2 (methyl-CpG-binding protein 2) gene. Over two decades of work established MeCP2 as a protein with pivotal roles in the regulation of the epigenome, neuronal physiology, synaptic maintenance, and behaviour. Given the genetic aetiology of RTT and the proof of concept of its reversal in a mouse model, considerable efforts have been made to design therapeutic approaches to re-express MeCP2. By being at the forefront of the development of innovative gene therapies, research on RTT is of paramount importance for the treatment of monogenic neurological diseases. Here we discuss the recent advances and challenges of promising genetic strategies for the treatment of RTT including gene replacement therapies, gene/RNA editing strategies, and reactivation of the silenced X chromosome.

BACKGROUND:The brain-derived neurotrophic factor (BDNF) Valine 66 to Methionine human polymorphism results in impaired activity-dependent BDNF release and has been linked to psychiatric disorders including depression and anxiety. We previously showed that male knock-in mice carrying the mouse Methionine homolog (Met68BDNF) exhibit excessive and compulsive alcohol drinking behaviors as compared to the wild-type Val68BDNF mice. OBJECTIVE:Here, we set out to determine the potential mechanism for the heightened and compulsive alcohol drinking phenotypes detected in Met68BDNF mice. RESULTS:We found that male, but not female Met68BDNF mice exhibit social anxiety-like behaviors. We further show that male Met68BDNF mice exhibit a preference for alcohol over social interaction. In contrast, alcohol place preference without an alternative social reward, is similar in male Met68BDNF and Val68BDNF mice. Since the Met68BDNF mice show social anxiety phenotypes, we tested whether alcohol reliefs anxiety similarly in Met68BDNF and Val68BDNF mice and found that male, but not female Met68BDNF mice are insensitive to the acute anxiolytic action of alcohol. Finally, we show that this acute tolerance to alcohol-dependent anxiolysis can be restored by overexpressing wild-type Val68BDNF in the ventral hippocampus (vHC) of Met68BDNF mice. CONCLUSIONS:Together, our results suggest that excessive alcohol drinking in the Met68BDNF may be attributed, in part, to heighted social anxiety and a lack of alcohol-dependent anxiolysis, a phenotype that is associated with malfunction of BDNF signaling in the vHC of male Met68BDNF mice.

Gene therapy represents a powerful therapeutic tool to treat diseased tissues and provide a durable and effective correction. The central nervous system (CNS) is the target of many gene therapy protocols, but its high complexity makes it one of the most difficult organs to reach, in part due to the blood-brain barrier that protects it from external threats. Focused ultrasound (FUS) coupled with microbubbles appears as a technological breakthrough to deliver therapeutic agents into the CNS. While most studies focus on a specific targeted area of the brain, the present work proposes to permeabilize the entire brain for gene therapy in several pathologies. Our results show that, after i.v. administration and FUS sonication in a raster scan manner, a self-complementary AAV9-CMV-GFP vector strongly and safely infected the whole brain of mice. An increase in vector DNA (19.8 times), GFP mRNA (16.4 times), and GFP protein levels (17.4 times) was measured in whole brain extracts of FUS-treated GFP injected mice compared to non-FUS GFP injected mice. In addition to this increase in GFP levels, on average, a 7.3-fold increase of infected cells in the cortex, hippocampus, and striatum was observed. No side effects were detected in the brain of treated mice. The combining of FUS and AAV-based gene delivery represents a significant improvement in the treatment of neurological genetic diseases.

SEE CORRESPONDING ARTICLE ON PAGE 959 The PI3-Kinase p110β Isoform Controls Severity of Cocaine-Induced Sequelae and Alters the Striatal TranscriptomeBiological PsychiatryVol. 89Issue 10PreviewThe PI3-kinase (PI3K) complex is a well-validated target for mitigating cocaine-elicited sequelae, but pan-PI3K inhibitors are not viable long-term treatment options. The PI3K complex is composed of p110 catalytic and regulatory subunits, which can be individually manipulated for therapeutic purposes. However, this possibility has largely not been explored in behavioral contexts. Full-Text PDF ErratumBiological PsychiatryVol. 90Issue 1PreviewErratum to: “PI3-Kinase p110β in Cortical Circuitries and Cocaine-Dependent Maladaptive Behaviors,” by Yann Ehinger (Biol Psychiatry 2021; 89:e55-e57); https://doi.org/10.1016/j.biopsych.2021.03.011 . Full-Text PDF

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent genetic cause of intellectual disability in girls, and there is currently no cure for the disease. We have previously shown that gene therapy using a self-complementary AAV9 viral vector expressing a codon-optimized Mecp2 version (AAV9-MCO) significantly improved symptoms and increased survival in male Mecp2-deficient mice. Here, we pursued our studies and investigated the safety and efficacy of long-term gene therapy in the genetically relevant RTT mouse model: the heterozygous (HET) Mecp2 deficient female mouse. These mice were injected with the AAV9-MCO vector through the tail vein and an array of behavioral tests was performed. At 16- and 30-weeks post-injection, this treatment was able to rescue apneas and improved the spontaneous locomotor deficits and circadian locomotor activity in Mecp2 HET mice treated with AAV9-MCO at a dose of 5 × 1011 vg/mouse.To examine whether a higher dose of vector could result in increased improvements, we injected Mecp2 HET mice with a higher MCO vector dose (1012 vg/mouse), which resulted in some severe, sometimes lethal, side effects. In order to confirm these effects, a new cohort of Mecp2 HET mice were administered increasing doses of MCO vector (1011, 5 × 1011 and 1012 vg/mouse). Again, two weeks after vector administration, some Mecp2 HET mice were found dead while others displayed severe side effects and had to be euthanized. These deleterious effects were not observed in Mecp2 HET mice injected with a high dose of AAV9-GFP and were directly proportionate to vector dosage (0, 23 or 54% mortality at an AAV9-MCO dose of 1011, 5 × 1011, 1012 vg/mouse, respectively), and no such lethality was observed in wild-type (WT) mice.In the Mecp2 HET mice treated with the high and medium AAV9-MCO doses, blood chemistry analysis and post-mortem histology showed liver damage with drastically elevated levels of liver transaminases and disorganized liver architecture. Apoptosis was confirmed by the presence of TUNEL- and cleaved-caspase 3-positive cells in the Mecp2 HET mice treated with the higher doses of AAV9-MCO. We then studied the involvement of the unfolded protein response (UPR) in triggering apoptosis since it can be activated by AAV vectors. Increased expression of the C/EBP homologous protein (CHOP), one of UPR downstream effectors, was confirmed in Mecp2 HET mice after vector administration.The toxic reaction seen in some treated mice indicates that, although gene therapy for RTT improved breathing deficits observed in Mecp2 HET mice, further studies are needed to better understand the underlying mechanisms and caution must be exercised before similar attempts are undertaken in female Rett patients.

Alcohol Use Disorder (AUD) affects a large portion of the population. Unfortunately, efficacious medications to treat the disease are limited. Studies in rodents suggest that mTORC1 plays a crucial role in mechanisms underlying phenotypes such as heavy alcohol intake, habit, and relapse. Thus, mTORC1 inhibitors, which are used in the clinic, are promising therapeutic agents to treat AUD. However, chronic inhibition of mTORC1 in the periphery produces undesirable side effects, which limit their potential use for the treatment of AUD. To overcome these limitations, we designed a binary drug strategy in which male mice were treated with the mTORC1 inhibitor RapaLink-1 together with a small molecule (RapaBlock) to protect mTORC1 activity in the periphery. We show that whereas RapaLink-1 administration blocked mTORC1 activation in the liver, RapaBlock abolished the inhibitory action of Rapalink-1. RapaBlock also prevented the adverse side effects produced by chronic inhibition of mTORC1. Importantly, co-administration of RapaLink-1 and RapaBlock inhibited alcohol-dependent mTORC1 activation in the nucleus accumbens and attenuated alcohol seeking and drinking.