The small G-protein Rac1 in the dorsomedial striatum promotes alcohol-dependent structural plasticity and goal-directed learning in mice.

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.

Paradoxical mTORC1-Dependent microRNA-mediated Translation Repression in the Nucleus Accumbens of Mice Consuming Alcohol Attenuates Glycolysis.

mTORC1 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 miR34a-5p. In parallel, we detected a paradoxical mTORC1-dependent repression of translation of transcripts including Aldolase A, an essential glycolytic enzyme. We found that miR34a-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.

The small G-protein Rac1 in the dorsomedial striatum promotes alcohol-dependent structural plasticity and goal-directed learning in mice.

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 male and female mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS, but not in the NAc and DLS of male mice. Chronic excessive alcohol intake does not activate Rac1 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. We infected 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 process. These results suggest that Rac1 in the DMS is an important contributor to adaptations that promote alcohol use disorder.

Voluntary alcohol binge-drinking in adolescent C57Bl6 mice induces delayed appearance of behavioural defects in both males and females.

Adolescence is a developmental period characterized by significant changes in brain architecture and behaviour. The immaturity of the adolescent brain is associated with heightened vulnerability to exogenous agents, including alcohol. Alcohol is the most consumed drug among teenagers, and binge-drinking during adolescence is a major public health concern. Studies have suggested that adolescent alcohol exposure may interfere with the maturation of frontal brain regions and lead to long-lasting behavioural consequences. In this study, by using a slightly modified version of the Drinking in the Dark paradigm, adolescent C57Bl6 mice reach high blood alcohol concentration after voluntary binge-drinking. In order to assess short- and long-term consequences of adolescent alcohol exposure (AAE), a battery of behavioural tests was performed during late adolescence and during adulthood. We showed that AAE had no short-term effect on young mice behaviour but rather increased anxiety- and depressive-like behaviours, as well as alcohol consumption during adulthood. Moreover, alcohol binge-drinking during adolescence dramatically decreased recognition memory performances and behavioural flexibility in both adult males and females. Furthermore, we showed that voluntary consumption of alcohol during adolescence did not trigger any major activation of the innate immune system in the prefrontal cortex. Together, our data suggest that voluntary alcohol binge-drinking in adolescent mice induces a delayed appearance of behavioural impairments in adulthood.

ATP-citrate lyase promotes axonal transport across species.

Microtubule (MT)-based transport is an evolutionary conserved process finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, primarily catalyzed by a vesicular pool of α-tubulin N-acetyltransferase 1 (Atat1), promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanism that controls Atat1 activity remains poorly understood. Here, we show that ATP-citrate lyase (Acly) is enriched in vesicles and provide Acetyl-Coenzyme-A (Acetyl-CoA) to Atat1. In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in a pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine light on the pathophysiological mechanisms underlying FD.

Differential correlation of serum BDNF and microRNA content in rats with rapid or late onset of heavy alcohol use.

Heavy alcohol use reduces the levels of the brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of rodents through the upregulation of microRNAs (miRs) targeting BDNF mRNA. In humans, an inverse correlation exists between circulating blood levels of BDNF and the severity of psychiatric disorders including alcohol abuse. Here, we set out to determine whether a history of heavy alcohol use produces comparable alterations in the blood of rats. We used an intermittent access to 20% alcohol using the two-bottle choice paradigm (IA20%2BC) and measured circulating levels of BDNF protein and miRs targeting BDNF in the serum of Long-Evans rats before and after 8 weeks of excessive alcohol intake. We observed that the drinking profile of heavy alcohol users is not unified, whereas 70% of the rats gradually escalate their alcohol intake (late onset), and 30% of alcohol users exhibit a very rapid onset of drinking (rapid onset). We found that serum BDNF levels are negatively correlated with alcohol intake in both rapid onset and late onset rats. In contrast, increased expression of the miRs targeting BDNF, miR30a-5p, miR-195-5p, miR191-5p and miR206-3p, was detected only in the rapid onset rats. Finally, we report that the alcohol-dependent molecular changes are not due to alterations in platelet number. Together, these data suggest that rats exhibit both late and rapid onset of alcohol intake. We further show that heavy alcohol use produces comparable changes in BDNF protein levels in both groups. However, circulating microRNAs are responsive to alcohol only in the rapid onset rats.

Protein Translation and Psychiatric Disorders.

Although historically research has focused on transcription as the central governor of protein expression, protein translation is now increasingly being recognized as a major factor for determining protein levels within cells. The central nervous system relies on efficient updating of the protein landscape. Thus, coordinated regulation of mRNA localization, initiation, or termination of translation is essential for proper brain function. In particular, dendritic protein synthesis plays a key role in synaptic plasticity underlying learning and memory as well as cognitive processes. Increasing evidence suggests that impaired mRNA translation is a common feature found in numerous psychiatric disorders. In this review, we describe how malfunction of translation contributes to development of psychiatric diseases, including schizophrenia, major depression, bipolar disorder, and addiction.

Proteostasis is essential during cochlear development for neuron survival and hair cell polarity.

Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

mTORC2 in the dorsomedial striatum of mice contributes to alcohol-dependent F-Actin polymerization, structural modifications, and consumption.

Actin is highly enriched at dendritic spines, and actin remodeling plays an essential role in structural plasticity. The mammalian target of rapamycin complex 2 (mTORC2) is a regulator of actin polymerization. Here, we report that alcohol consumption increases F-actin content in the dorsomedial striatum (DMS) of mice, thereby altering dendritic spine morphology in a mechanism that requires mTORC2. Specifically, we found that excessive alcohol consumption increases mTORC2 activity in the DMS, and that knockdown of Rictor, an essential component of mTORC2 signaling, reduces actin polymerization, and attenuates the alcohol-dependent alterations in spine head size and the number of mushroom spines. Finally, we show that knockdown of Rictor in the DMS reduces alcohol consumption, whereas intra-DMS infusion of the mTORC2 activator, A-443654, increases alcohol intake. Together, these results suggest that mTORC2 in the DMS facilitates the formation of F-actin, which in turn induces changes in spine structure to promote and/or maintain excessive alcohol intake.

Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and Reward.

The mammalian target of rapamycin complex 1 (mTORC1), a transducer of local dendritic translation, participates in learning and memory processes as well as in mechanisms underlying alcohol-drinking behaviors. Using an unbiased RNA-seq approach, we identified Prosapip1 as a novel downstream target of mTORC1 whose translation and consequent synaptic protein expression are increased in the nucleus accumbens (NAc) of mice excessively consuming alcohol. We demonstrate that alcohol-dependent increases in Prosapip1 levels promote the formation of actin filaments, leading to changes in dendritic spine morphology of NAc medium spiny neurons (MSNs). We further demonstrate that Prosapip1 is required for alcohol-dependent synaptic localization of GluA2 lacking AMPA receptors in NAc shell MSNs. Finally, we present data implicating Prosapip1 in mechanisms underlying alcohol self-administration and reward. Together, these data suggest that Prosapip1 in the NAc is a molecular transducer of structural and synaptic alterations that drive and/or maintain excessive alcohol use.

Loss of Elp3 Impairs the Acetylation and Distribution of Connexin-43 in the Developing Cerebral Cortex.

The Elongator complex is required for proper development of the cerebral cortex. Interfering with its activity in vivo delays the migration of postmitotic projection neurons, at least through a defective α-tubulin acetylation. However, this complex is already expressed by cortical progenitors where it may regulate the early steps of migration by targeting additional proteins. Here we report that connexin-43 (Cx43), which is strongly expressed by cortical progenitors and whose depletion impairs projection neuron migration, requires Elongator expression for its proper acetylation. Indeed, we show that Cx43 acetylation is reduced in the cortex of Elp3cKO embryos, as well as in a neuroblastoma cell line depleted of Elp1 expression, suggesting that Cx43 acetylation requires Elongator in different cellular contexts. Moreover, we show that histones deacetylase 6 (HDAC6) is a deacetylase of Cx43. Finally, we report that acetylation of Cx43 regulates its membrane distribution in apical progenitors of the cerebral cortex.

Region specific activation of the AKT and mTORC1 pathway in response to excessive alcohol intake in rodents.

We previously reported that the kinase AKT is activated in the nucleus accumbens (NAc) of rodents in response to excessive consumption of alcohol. One of the important downstream targets of AKT is the mammalian Target Of Rapamycin in Complex 1 (mTORC1), which was also activated by alcohol intake. mTORC1 controls dendritic protein translation, and we showed that the mTORC1-dependent translational machinery is activated in the NAc in response to alcohol intake. Importantly, systemic or intra-NAc inhibition of the AKT/mTORC1 pathway attenuated alcohol-drinking behaviors. Here, we mapped the activation patterns of AKT and mTORC1 in corticostriatal regions of rodents consuming large amounts of alcohol. We found that the activation of AKT and mTORC1 in response to cycles of binge drinking of 20 percent alcohol was centered in the NAc shell. Both kinases were not activated in the dorsolateral striatum (DLS); however, AKT, but not mTORC1, was activated in the dorsomedial striatum (DMS) of mice but not rats. Interestingly, excessive intake of alcohol produced a selective activation of the AKT/mTORC1 pathway in the orbitofrontal cortex (OFC), which was not observed in medial prefrontal cortex (mPFC). Furthermore, this signaling pathway was not activated in the NAc shell or OFC of rats consuming moderate amounts of alcohol nor was it activated in rats consuming sucrose. Together, our results suggest that excessive alcohol intake produces a brain region selective activation of the AKT/mTORC1 pathway, which is likely to contribute to NAc shell and OFC-dependent mechanisms that underlie the development and maintenance of alcohol drinking behavior.

Emerging Roles for the Unfolded Protein Response in the Developing Nervous System.

The unfolded protein response (UPR) is a homeostatic signaling pathway triggered by protein misfolding in the endoplasmic reticulum (ER). Beyond its protective role, it plays important functions during normal development in response to elevated demand for protein folding. Several UPR effectors show dynamic temporal and spatial expression patterns that correlate with milestones of the central nervous system (CNS) development. Here, we discuss recent studies suggesting that a dynamic regulation of UPR supports generation, maturation, and maintenance of differentiated neurons in the CNS. We further highlight studies supporting a developmental vulnerability of CNS to UPR dysregulation, which underlies neurodevelopmental disorders. We believe that a better understanding of UPR functions may provide novel opportunities for therapeutic strategies to fight ER/UPR-associated human neurological disorders.

The First Alcohol Drink Triggers mTORC1-Dependent Synaptic Plasticity in Nucleus Accumbens Dopamine D1 Receptor Neurons.

Early binge-like alcohol drinking may promote the development of hazardous intake. However, the enduring cellular alterations following the first experience with alcohol consumption are not fully understood. We found that the first binge-drinking alcohol session produced enduring enhancement of excitatory synaptic transmission onto dopamine D1 receptor-expressing neurons (D1+ neurons) in the nucleus accumbens (NAc) shell but not the core in mice, which required D1 receptors (D1Rs) and mechanistic target of rapamycin complex 1 (mTORC1). Furthermore, inhibition of mTORC1 activity during the first alcohol drinking session reduced alcohol consumption and preference of a subsequent drinking session. mTORC1 is critically involved in RNA-to-protein translation, and we found that the first alcohol session rapidly activated mTORC1 in NAc shell D1+ neurons and increased synaptic expression of the AMPAR subunit GluA1 and the scaffolding protein Homer. Finally, D1R stimulation alone was sufficient to activate mTORC1 in the NAc to promote mTORC1-dependent translation of the synaptic proteins GluA1 and Homer. Together, our results indicate that the first alcohol drinking session induces synaptic plasticity in NAc D1+ neurons via enhanced mTORC1-dependent translation of proteins involved in excitatory synaptic transmission that in turn drives the reinforcement learning associated with the first alcohol experience. Thus, the alcohol-dependent D1R/mTORC1-mediated increase in synaptic function in the NAc may reflect a neural imprint of alcohol's reinforcing properties, which could promote subsequent alcohol intake. Significance statement: Consuming alcohol for the first time is a learning event that drives further drinking. Here, we identified a mechanism that may underlie the reinforcing learning associated with the initial alcohol experience. We show that the first alcohol experience induces a persistent enhancement of excitatory synaptic transmission on NAc shell D1+ neurons, which is dependent on D1R and mTORC1. We also find that mTORC1 is necessary for the sustained alcohol consumption and preference across the initial drinking sessions. The first alcohol binge activates mTORC1 in NAc D1+ neurons and increases levels of synaptic proteins involved in glutamatergic signaling. Thus, the D1R/mTORC1-dependent plasticity following the first alcohol exposure may be a critical cellular component of reinforcement learning.

A Dynamic Unfolded Protein Response Contributes to the Control of Cortical Neurogenesis.

The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs), whose asymmetric division gives birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Here we show that interfering with codon translation speed triggers ER stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a contribution of UPR to cell fate acquisition during mammalian brain development.

Progenitor genealogy in the developing cerebral cortex.

The mammalian cerebral cortex is characterized by a complex histological organization that reflects the spatio-temporal stratifications of related stem and neural progenitor cells, which are responsible for the generation of distinct glial and neuronal subtypes during development. Some work has been done to shed light on the existing filiations between these progenitors as well as their respective contribution to cortical neurogenesis. The aim of the present review is to summarize the current views of progenitor hierarchy and relationship in the developing cortex and to further discuss future research directions that would help us to understand the molecular and cellular regulating mechanisms involved in cerebral corticogenesis.

MicroRNA targeting of CoREST controls polarization of migrating cortical neurons.

The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. We show that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex, thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, our work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis.