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Drug addiction develops due to brain-wide plasticity within neuronal ensembles, mediated by dynamic gene expression. Though the most common approach to identify such ensembles relies on immediate early gene expression, little is known of how the activity of these genes is linked to modified behavior observed following repeated drug exposure. To address this gap, we present a broad-to-specific approach, beginning with a comprehensive investigation of brain-wide cocaine-driven gene expression, through the description of dynamic spatial patterns of gene induction in subregions of the striatum, and finally address functionality of region-specific gene induction in the development of cocaine preference. Our findings reveal differential cell-type specific dynamic transcriptional recruitment patterns within two subdomains of the dorsal striatum following repeated cocaine exposure. Furthermore, we demonstrate that induction of the IEG Egr2 in the ventrolateral striatum, as well as the cells within which it is expressed, are required for the development of cocaine seeking. The human brain is ever changing, constantly rewiring itself in response to new experiences, knowledge or information from the environment. Addictive drugs such as cocaine can hijack the genetic mechanisms responsible for this plasticity, creating dangerous, obsessive drug-seeking and consuming behaviors. Cocaine-induced plasticity is difficult to apprehend, however, as brain regions or even cell populations can react differently to the compound. For instance, sub-regions in the striatum – the brain area that responds to rewards and helps to plan movement – show distinct responses during progressive exposure to cocaine. And while researchers know that the drug immediately changes how neurons switch certain genes on and off, it is still unclear how these genetic modifications later affect behavior. Mukherjee, Gonzales et al. explored these questions at different scales, first focusing on how progressive cocaine exposure changed the way various gene programs were activated across the entire brain. This revealed that programs in the striatum were the most affected by the drug. Examining this region more closely showed that cocaine switches on genes in specific ‘spiny projection’ neuron populations, depending on where these cells are located and the drug history of the mouse. Finally, Mukherjee, Gonzales et al. used genetically modified mice to piece together cocaine exposure, genetic changes and modifications in behavior. These experiments revealed that the drive to seek cocaine depended on activation of the Egr2 gene in populations of spiny projection neurons in a specific sub-region of the striatum. The gene, which codes for a protein that regulates how genes are switched on and off, was itself strongly activated by cocaine intake. Cocaine addiction can have devastating consequences for individuals. Grasping how this drug alters the brain could pave the way for new treatments, while also providing information on the basic mechanisms underlying brain plasticity.

It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory. Can we tell what important event a mouse – or even a person – has recently experienced? The current experience of an individual can be inferred from brain imaging experiments. However, along with changing brain activity, such an experience also switches on gene activity throughout the brain. This enables neurons to produce the proteins required to form a long-term memory of the experience. Do distinct, memorable experiences trigger unique signatures of gene activity? To answer this question, Mukherjee, Ignatowska-Jankowska, Itskovits et al. exposed mice to a variety of experiences. Some were unpleasant and induced aversion; for example, the mouse may have felt nauseous or experienced brief pain and fear. Other experiences, such as when the mouse drank sugary water, received food or was injected with cocaine, were rewarding. Each of the experiences led to the activation of unique combinations of genes in different regions of the brain. Analysing a subset of the activated genes in various brain regions led to the identification of unique and reliable gene expression signatures of experience. These signatures allowed the recent experience of mice to be decoded with nearly 100% accuracy. While these unique signatures can distinguish between recent experiences, experiences that share common features do trigger overlapping patterns of gene activation. For example, negative experiences – but not positive or neutral ones – activated similar patterns of genes in a brain region called the amygdala. In contrast, repeated rewarding experiences induced a distinct gene activity pattern that was most pronounced as increased activity in part of the brain called the frontal cortex. These findings increase our understanding of how the brain represents information. The approach described in the paper provides a strategy to measure the changes in the brain that occur when information is encoded for long-term storage. This measure could also be useful during drug development, revealing how new drug compounds affect the brain, as well as providing an objective measure of the subjective experience of an individual. For example, substances that trigger similar patterns of gene activation to addictive drugs may themselves be addictive. On the other hand, substances that induce similar activity patterns to known medications could also have similar therapeutic properties.

Blood transcriptomic signatures for Parkinson’s disease (PD) diagnosis have failed to integrate into clinical practice despite decades of translational efforts. We evaluated the classification performance of 13 published coding RNA-based signatures using both a large public dataset and data we collected prospectively in a controlled clinical study of levodopa-naïve patients and healthy controls. Our results show that gene overlap between signatures is low but significant (2.7%, p  < 0.001) and enriched for lipid metabolism genes. Most signatures (10/13) remained significant when tested on the Parkinson’s Progression Markers Initiative (PPMI) dataset, though with lower classification performance than previously reported (median AUC: 59.7%). Performance improved for GBA1 -associated PD. Rigorous standardization of clinical and environmental parameters in our prospective study (30 participants) failed to improve transcriptome-based classification. We conclude that while the search for a universal blood-based PD transcriptome may elucidate disease pathophysiology, its clinical utility is inherently limited.

While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche.

Rationale and objectives Social factors play a critical role in human drug addiction, and humans often consume drugs together with their peers. In contrast, in traditional animal models of addiction, rodents consume or self-administer the drug in their homecage or operant self-administration chambers while isolated from their peers. Here, we describe HOMECAGE (“Home-cage Observation and Measurement for Experimental Control and Analysis in a Group-housed Environment”), a translationally relevant method for studying oral opioid self-administration in mice. This setting reduces experimental confounds introduced by social isolation or interaction with the experimenter. Methods We have developed HOMECAGE, a method in which mice are group-housed and individually monitored for their consumption of a drug vs. a reference liquid. Results Mice in HOMECAGE preserve naturalistic aspects of behavior, including social interactions and circadian activity. The mice showed a preference for fentanyl and escalated their fentanyl intake over time. Mice preferred to consume fentanyl in bouts during the dark cycle. Mice entrained to the reinforcement schedule of the task, optimizing their pokes to obtain fentanyl rewards, and maintained responding for fentanyl under a progressive ratio schedule. HOMECAGE also enabled the detection of cage-specific and individual-specific behavior patterns and allowed the identification of differences in fentanyl consumption between co-housed control and experimental mice. Conclusions HOMECAGE serves as a valuable procedure for translationally relevant studies on oral opioid intake under conditions that more closely mimic the human condition. The method enables naturalistic investigation of factors contributing to opioid addiction-related behaviors and can be used to identify novel treatments.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

Qiu Y, Robinson D, Pretlow TG, Kung HJ (1998) Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3'-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells. Abassi YA, Rehn M, Ekman N, Alitalo K, Vuori K (2003) p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration. Lee LF, Guan J, Qiu Y, Kung HJ (2001) Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase. (2002) Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis.

While protease-activated-receptor 1 (PAR.sub.1) plays a central role in tumor progression, little is known about the cell signaling involved. We show here the impact of PAR.sub.1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR.sub.1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR.sub.1 C-tail did not prevent Shc-PAR.sub.1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR.sub.1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR.sub.1 -induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR.sub.1 -C-tail, hPar1 oncogenic properties are abrogated. This is the first demonstration that a cytoplasmic portion of the PAR.sub.1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR.sub.1 -associating region in the breast cancer signaling niche.