An automated group-housed oral fentanyl self-administration method in mice.

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.

Management and outcome of 500 multiple myeloma patients treated for first relapse outside clinical studies.

Treatment options for multiple myeloma (MM) at 1st relapse are expanding. The current study compared common 2nd line regimens administered in a real-world setting. MM patients registered in Maccabi health care services and treated with second line therapy during 2014-2020 were evaluated, analyzing factors affecting time to third line therapy (TT3T). The study included 500 MM patients, previously treated with proteasome inhibitor (PI)-based induction. Median age at second line treatment was 68.5 years (IQR: 61.6-76.4). Most patients received a triplet based induction composed of PI (n = 471, 94.2%), with (n = 71) or without IMID (n = 400), followed by second line treatment composed of lenalidomide-dexamethasone (RD) (n = 225, 45%) or lenalidomide-dexamethasone-daratumumab (RD-Dara (n = 104, 20.8%)). Multivariable analysis confirmed treatment type (RD-Dara vs. IMID) to be associated with a lower risk to progress to third line therapy; (HR = 0.5, 95% CI 0.3-0.86, p = 0.012). Within a median follow-up period of 22.5 months (intraquartile range 11.1-39.4 m), median TT3T was not reached in patients receiving RD-Dara vs. 32.4 months (95% CI 18.0-46.8 m) with IMID, 18 months (95% CI 10.4-25.6 m) with IMID-PI and 12.1 months (95% CI 5.6-18.7 m) with PI-based regimen. In contrast, PI vs. IMID-based therapy and increased body weight were associated with a higher likelihood of progression (HR = 2.56 (95% CI 1.49-4.42); HR = 1.43, (95% CI 0.96-2.14), p = 0.08). To conclude, second line therapy with RD-Dara was associated with a significantly longer TT3T compared with IMID-based regimen, longer than obtained with PI-IMID and PI-based regimens, in patients treated outside clinical studies and previously exposed to bortezomib.

Claustral neurons projecting to frontal cortex restrict opioid consumption.

The synthetic opioid fentanyl is a major contributor to the current opioid addiction crisis. We report that claustral neurons projecting to the frontal cortex limit oral fentanyl self-administration in mice. We found that fentanyl transcriptionally activates frontal-projecting claustrum neurons. These neurons also exhibit a unique suppression of Ca2+ activity upon initiation of bouts of fentanyl consumption. Optogenetic stimulation of frontal-projecting claustral neurons, intervening in this suppression, decreased bouts of fentanyl consumption. In contrast, constitutive inhibition of frontal-projecting claustral neurons in the context of a novel, group-housed self-administration procedure increased fentanyl bout consumption. This same manipulation also sensitized conditioned-place preference for fentanyl and enhanced the representation of fentanyl experience in the frontal cortex. Together, our results indicate that claustrum neurons exert inhibitory control over frontal cortical neurons to restrict oral fentanyl intake. Upregulation of activity in the claustro-frontal projection may be a promising strategy for reducing human opioid addiction.

Claustral Neurons Projecting to Frontal Cortex Mediate Contextual Association of Reward.

The claustrum is a small nucleus, exhibiting vast reciprocal connectivity with cortical, subcortical, and midbrain regions. Recent studies, including ours, implicate the claustrum in salience detection and attention. In the current study, we develop an iterative functional investigation of the claustrum, guided by quantitative spatial transcriptional analysis. Using this approach, we identify a circuit involving dopamine-receptor expressing claustral neurons projecting to frontal cortex necessary for context association of reward. We describe the recruitment of claustral neurons by cocaine and their role in drug sensitization. In order to characterize the circuit within which these neurons are embedded, we apply chemo- and opto-genetic manipulation of increasingly specified claustral subpopulations. This strategy resolves the role of a defined network of claustrum neurons expressing dopamine D1 receptors and projecting to frontal cortex in the acquisition of cocaine conditioned-place preference and real-time optogenetic conditioned-place preference. In sum, our results suggest a role for a claustrum-to-frontal cortex circuit in the attribution of incentive salience, allocating attention to reward-related contextual cues.

The Claustrum Supports Resilience to Distraction.

A barrage of information constantly assaults our senses, of which only a fraction is relevant at any given point in time. However, the neural circuitry supporting the suppression of irrelevant sensory distractors is not completely understood. The claustrum, a circuit hub with vast cortical connectivity, is an intriguing brain structure, whose restrictive anatomy, thin and elongated, has precluded functional investigation. Here, we describe the use of Egr2-CRE mice to access genetically defined claustral neurons. Utilizing conditional viruses for anterograde axonal labeling and retrograde trans-synaptic tracing, we validated this transgenic model for accessing the claustrum and extended the known repertoire of claustral input/output connectivity. Addressing the function of the claustrum, we inactivated CLEgr2+ neurons, chronically as well as acutely, in mice performing an automated two-alternative forced-choice behavioral task. Strikingly, inhibition of CLEgr2+ neurons did not significantly impact task performance under varying delay times and cue durations, but revealed a selective role for the claustrum in supporting performance in the presence of an irrelevant auditory distractor. Further investigation of behavior, in the naturalistic maternal pup-retrieval task, replicated the result of sensitization to an auditory distractor following inhibition of CLEgr2+ neurons. Initiating investigation into the underlying mechanism, we found that activation of CLEgr2+ neurons modulated cortical sensory processing, suppressing tone representation in the auditory cortex. This functional study, utilizing selective genetic access, implicates the claustrum in supporting resilience to distraction, a fundamental aspect of attention.

Thymospheres Are Formed by Mesenchymal Cells with the Potential to Generate Adipocytes, but Not Epithelial Cells.

Evidence suggests that a stem-cell-driven differentiation hierarchy maintains the dynamic thymic epithelial cell (TEC) network that governs T lymphocyte development. The identification of TEC stem/progenitor cells has been a major focus in the field, and several candidates with contrasting phenotypes have been described. We sought to determine the provenance and function of the only population reported to exhibit TEC stem cell properties in the adult, a Foxn1- EpCAM- cell that generates so-called thymospheres. We provide evidence that the thymosphere-forming cell (TSFC) is not a TEC stem cell but can incorporate bystander TECs into thymospheres, providing an explanation for the epithelial activity ascribed to these structures. TSFCs were found to share a phenotype, transcriptional profile, and developmental origin with thymic fibroblasts and can generate adipocytes. In summary, this study redefines the nature of bipotent TEC stem/progenitor cells in the adult thymus and highlights a potentially important mesenchymal progenitor population.

Mapping synaptic cortico-claustral connectivity in the mouse.

The claustrum is an intriguing brain structure, featuring the highest connectivity per regional volume in the brain. It is a thin and elongated structure enclosed between the striatum and the insular cortex, with widespread reciprocal connections with the sensory modalities and prefrontal cortices. Retinotopic and somatotopic organizations have been described in the claustrum, and anatomical studies in cats, monkeys, and rats have demonstrated topographic organization of cortico-claustral connections. In this study we mapped the projections from cortical modalities (visual, auditory, somatosensory, motor, and olfactory), and prefrontal regions (anterior cingulate cortex and orbitofrontal cortex) to the claustrum in mice. Utilizing expression of a virally encoded synaptic anterograde tracer, AAV-SynaptoTag, followed by 3D reconstruction of the cortical projections, we performed a comprehensive study of the organization of these projections within the mouse claustrum. Our results clearly demonstrate a dorsoventral laminar organization of projections from the sensory cortices to the claustrum, whereas frontal inputs are more extensive and overlap with the inputs from the sensory cortices. In addition, we find evidence supporting a core/shell organization of the claustrum. We propose that the overlap between the frontal inputs and the inputs from the sensory modalities may underlie executive regulation of the communication between the claustrum and the cortical modalities. J. Comp. Neurol. 525:1381-1402, 2017. © 2016 Wiley Periodicals, Inc.

p53 orchestrates between normal differentiation and cancer.

During recent years, it is becoming more and more evident that there is a tight connection between abnormal differentiation processes and cancer. While cancer and stem cells are very different, especially in terms of maintaining genomic integrity, these cell types also share many similar properties. In this review, we aim to provide an over-view of the roles of the key tumor suppressor, p53, in regulating normal differentiation and function of both stem cells and adult cells. When these functions are disrupted, undifferentiated cells may become transformed. Understanding the function of p53 in stem cells and its role in maintaining the balance between differentiation and malignant transformation can help shed light on cancer initiation and propagation, and hopefully also on cancer prevention and therapy.

p53 in liver pathologies-taking the good with the bad.

The distinct physiology of the liver makes it a unique ground with respect to its cross talk with p53, the "guardian of the genome." The stressful environment in the liver frequently leads to the activation of p53, which is associated with alterations in metabolic pathways and induction of apoptosis. The latter serves as a mechanism that controls the deposal of DNA-damaged cells. However, accentuated apoptosis may eventually lead to liver pathologies, mainly steatosis, which can develop into a more severe disease such as steatohepatitis, fibrosis, and cirrhosis. These pathologies, together with other apoptosis outcome such as chronic inflammation, may pave the way toward cancer development. In addition to this unique scenario that connects the ongoing response of wild-type (WT) p53 to stress and cancer development, hepatocarcinoma may develop in other well-described mechanisms involving p53. One such example is hepatitis virus-induced liver cancer whereby p53 is inactivated upon the binding of a specific viral protein, leading to the loss of its tumor suppressive activity. Furthermore, the accumulations of carcinogens such as aflatoxin were shown to yield an oncogenic mutated p53 protein. In this review, we will demonstrate the diverse activities of p53 in the liver. Interestingly, some of these activities may protect the liver from cancer in the short term, yet in the long term, p53 may lead to malignant transformation. A better understanding of the complex clinical outcome of p53 function in the liver may shed light on future therapies.

Rescue of embryonic stem cells from cellular transformation by proteomic stabilization of mutant p53 and conversion into WT conformation.

p53 is a well-known tumor suppressor that is mutated in over 50% of human cancers. These mutations were shown to exhibit gain of oncogenic function compared with the deletion of the gene. Additionally, p53 has fundamental roles in differentiation and development; nevertheless, mutant p53 mice are viable and develop malignant tumors only on adulthood. We set out to reveal the mechanisms by which embryos are protected from mutant p53-induced transformation using ES cells (ESCs) that express a conformational mutant of p53. We found that, despite harboring mutant p53, the ESCs remain pluripotent and benign and have relatively normal karyotype compared with ESCs knocked out for p53. Additionally, using high-content RNA sequencing, we show that p53 is transcriptionally active in response to DNA damage in mutant ESCs and elevates p53 target genes, such as p21 and btg2. We also show that the conformation of mutant p53 protein in ESCs is stabilized to a WT conformation. Through MS-based interactome analyses, we identified a network of proteins, including the CCT complex, USP7, Aurora kinase, Nedd4, and Trim24, that bind mutant p53 and may shift its conformation to a WT form. We propose this conformational shift as a novel mechanism of maintenance of genomic integrity, despite p53 mutation. Harnessing the ability of these protein interactors to transform the oncogenic mutant p53 to the tumor suppressor WT form can be the basis for future development of p53-targeted cancer therapy.

Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4 by activating p53.

Cytochrome P450 (P450) enzymes are abundantly expressed in the human liver where they hydroxylate organic substrates. In a microarray screen performed in human liver cells, we found a group of eleven P450 genes whose expression was induced by p53 (CYP3A4, CYP3A43, CYP3A5, CYP3A7, CYP4F2, CYP4F3, CYP4F11, CYP4F12, CYP19A1, CYP21A2 and CYP24A1). The mode of regulation of four representative genes (CYP3A4, CYP3A7, CYP4F2 and CYP4F3) was further characterized. The genes were induced in a p53-dependent manner in HepG2 and Huh6 cells (both are cancer-derived human liver cells) and in primary liver cells isolated from human donors. Furthermore, p53 was found to bind to p53-responsive elements in the genes' DNA-regulatory regions and to enhance their transcription in a reporter gene assay. Importantly, when p53 was activated following the administration of either of three different anticancer chemotherapeutic agents (cisplatin, etoposide or doxorubicin), it was able to induce CYP3A genes, which are the main factors in systemic clearance of these agents. Finally, the p53-dependent induction of P450 genes following either Nutlin or chemotherapy treatment led to enhanced P450 enzymatic activity. Thus, in addition to the well-established role of p53 at the tumor site, our data unravels a novel function of hepatic p53 in inducing P450 enzymes and position p53 as a major factor in the hepatic response to xenobiotic and metabolic signals. Importantly, this study reveals a novel pathway for the induction of CYP3As by their substrates through p53, warranting the need for careful consideration when designing systemically administered chemotherapeutic regimens.

p53, a novel regulator of lipid metabolism pathways.

BACKGROUND & AIMS: In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor. METHODS: Analysis of gene expression following alteration of p53 status in several human- and mouse-derived cells using microarray analysis, quantitative real-time PCR, chromatin immunoprecipitation, and reporter gene assays. A functional assay was performed to determine lipid transfer activity. RESULTS: We identified a novel role for the p53 protein in regulating lipid and lipoprotein metabolism, a process not yet conceived as related to p53, which is known mainly for its tumor suppressive functions. We revealed a group of 341 genes whose expression was induced by p53 in the liver-derived cell line HepG2. Twenty of these genes encode proteins involved in many aspects of lipid homeostasis. The mode of regulation of three representative genes (Pltp, Abca12, and Cel) was further characterized. In addition to HepG2, the genes were induced following activation of p53 in human primary hepatic cells isolated from liver donors. p53-dependent regulation of these genes was evident in other cell types namely Hep3B cells, mouse hepatocytes, and fibroblasts. Furthermore, p53 was found to bind to the genes' promoters in designated p53 responsive elements and thereby increase transcription. Importantly, p53 augmented the activity of secreted PLTP, which plays a major role in lipoprotein biology and atherosclerosis pathology. CONCLUSIONS: These findings expose another facet of p53 functions unrelated to tumor suppression and render it a novel regulator of hepatic lipid metabolism and consequently of systemic lipid homeostasis and atherosclerosis development.

Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis.

Inactivation of the p53 tumor suppressor is a frequent event in tumorigenesis. In most cases, the p53 gene is mutated, giving rise to a stable mutant protein whose accumulation is regarded as a hallmark of cancer cells. Mutant p53 proteins not only lose their tumor suppressive activities but often gain additional oncogenic functions that endow cells with growth and survival advantages. Interestingly, mutations in the p53 gene were shown to occur at different phases of the multistep process of malignant transformation, thus contributing differentially to tumor initiation, promotion, aggressiveness, and metastasis. Here, the authors review the different studies on the involvement of p53 inactivation at various stages of tumorigenesis and highlight the specific contribution of p53 mutations at each phase of cancer progression.

Mutant p53 facilitates somatic cell reprogramming and augments the malignant potential of reprogrammed cells.

p53 deficiency enhances the efficiency of somatic cell reprogramming to a pluripotent state. As p53 is usually mutated in human tumors and many mutated forms of p53 gain novel activities, we studied the influence of mutant p53 (mut-p53) on somatic cell reprogramming. Our data indicate a novel gain of function (GOF) property for mut-p53, which markedly enhanced the efficiency of the reprogramming process compared with p53 deficiency. Importantly, this novel activity of mut-p53 induced alterations in the characteristics of the reprogrammed cells. Although p53 knockout (KO) cells reprogrammed with only Oct4 and Sox2 maintained their pluripotent capacity in vivo, reprogrammed cells expressing mutant p53 lost this capability and gave rise to malignant tumors. This novel GOF of mut-p53 is not attributed to its effect on proliferation, as both p53 KO and mut-p53 cells displayed similar proliferation rates. In addition, we demonstrate an oncogenic activity of Klf4, as its overexpression in either p53 KO or mut-p53 cells induced aggressive tumors. Overall, our data show that reprogrammed cells with the capacity to differentiate into the three germ layers in vitro can form malignant tumors, suggesting that in genetically unstable cells, such as those in which p53 is mutated, reprogramming may result in the generation of cells with malignant tumor-forming potential.

p53 is balancing development, differentiation and de-differentiation to assure cancer prevention.

Many of the roles played by the tumor suppressor p53 in restraining cancer initiation and progression are well established. These include the ability of p53 to induce cell-cycle arrest, DNA repair, senescence and apoptosis. In addition, during the 30 years of p53 research, numerous studies have implicated p53 in the regulation of differentiation and developmental pathways. Here, we summarize the data on these relatively less-characterized functions of p53, including its involvement in embryogenesis and various differentiation programs, as well as its function in restraining de-differentiation of mature somatic cells. Besides the well-known functions of p53 as a cell-cycle regulator and a mediator of apoptosis, both coincide with differentiation processes, p53 was shown to exert its effects on various differentiation programs via direct regulation of specific key factors controlling these programs. The complex regulation by p53, which acts to suppress or to induce differentiation, is mainly the result of the specific cell type and fate. We argue that regulation of differentiation is pivotal for the tumor-suppressive activity of p53, which act to maintain the proper cellular state, preventing improper maturation or reprogramming. This conclusion is further supporting the notion that aberrant differentiation is associated with malignant transformation.

p53 Regulates the Ras circuit to inhibit the expression of a cancer-related gene signature by various molecular pathways.

In this study, we focus on the analysis of a previously identified cancer-related gene signature (CGS) that underlies the cross talk between the p53 tumor suppressor and Ras oncogene. CGS consists of a large number of known Ras downstream target genes that were synergistically upregulated by wild-type p53 loss and oncogenic H-Ras(G12V) expression. Here we show that CGS expression strongly correlates with malignancy. In an attempt to elucidate the molecular mechanisms underling the cooperation between p53 loss and oncogenic H-Ras(G12V), we identified distinguished pathways that may account for the regulation of the expression of the CGS. By knocking-down p53 or by expressing mutant p53, we revealed that p53 exerts its negative effect by at least two mechanisms mediated by its targets B-cell translocation gene 2 (BTG2) and activating transcription factor 3 (ATF3). Whereas BTG2 binds H-Ras(G12V) and represses its activity by reducing its GTP loading state, which in turn causes a reduction in CGS expression, ATF3 binds directly to the CGS promoters following p53 stabilization and represses their expression. This study further elucidates the molecular loop between p53 and Ras in the transformation process.