Loss of PTEN promotes formation of signaling-capable clathrin-coated pits.

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.

Cyclooxygenase-2 inhibition prevents stress induced amygdala activation and anxiety-like behavior.

Stress is a major risk factor for the development and exacerbation of mood and anxiety disorders, and recent studies have suggested inflammatory contributions to the pathogenesis of depression. Interestingly, pharmacological inhibition of cyclooxygenase-2 (COX-2) has shown promise in the treatment of affective disorders in small scale clinical studies; however, the mechanisms by which COX-2 inhibition affects behavioral domains relevant to affective disorders are not well understood. Here, we examined the effects of pharmacological inhibition of COX-2 with the highly selective inhibitor Lumiracoxib (LMX) on anxiety-like behavior and in vivo basolateral amygdala (BLA) neural activity in response to acute restraint stress exposure. In male mice, pretreatment with LMX prevented the increase in BLA calcium transients induced by restraint stress and prevented anxiogenic behavior seen after restraint stress exposure. Specifically, acute injection of LMX 5 mg kg-1 reduced anxiety-like behavior in the light-dark box (LD) and elevated-zero maze (EZM). In addition, in vivo fiber photometry studies showed that acute stress increased calcium transients and the predicted action potential frequency of BLA neurons, which was also normalized by acute LMX pretreatment. These findings indicate pharmacological inhibition of COX-2 can prevent acute stress-induced increase in BLA cellular activity and anxiety-like behavior and provides insights into the neural mechanisms by which COX-2 inhibition could affect anxiety domain symptoms in patients with affective disorders.

Operant ethanol self-administration behaviors do not predict sex differences in continuous access home cage drinking.

Understanding sex differences in disease prevalence is critical to public health, particularly in the context of alcohol use disorder (AUD). The goal of this study was to understand sex differences in ethanol drinking behavior and define the precise conditions under which sex differences emerge. Consistent with prior work, C57BL/6J females drank more than males under continuous access two-bottle choice conditions. However, using ethanol self-administration - where an operant response results in access to an ethanol sipper for a fixed time period - we found no sex differences in operant response rates or ethanol consumption (volume per body weight consumed, as well as lick behavior). This remained true across a wide range of parameters including acquisition, when the ethanol sipper access period was manipulated, and when the concentration of the ethanol available was scaled. The only sex differences observed were in total ethanol consumption, which was explained by differences in body weight between males and females, rather than by sex differences in motivation to drink. Using dimensionality reduction approaches, we found that drinking behavior in the operant context did not cluster by sex, but rather clustered by high and low drinking phenotypes. Interestingly, these high and low drinking phenotypes in the operant context showed no correlation with those same categorizations in the home cage context within the same animals. These data underscore the complexity of sex differences in ethanol consumption, highlighting the important role that drinking conditions/context plays in the expression of these differences.

PREFRONTAL CORRELATES OF FEAR GENERALIZATION DURING ENDOCANNABINOID DEPLETION.

Maladaptive fear generalization is one of the hallmarks of trauma-related disorders. The endocannabinoid 2-arachidonoylglycerol (2-AG) is crucial for modulating anxiety, fear, and stress adaptation but its role in balancing fear discrimination versus generalization is not known. To address this, we used a combination of plasma endocannabinoid measurement and neuroimaging from a childhood maltreatment exposed and non-exposed mixed population combined with human and rodent fear conditioning models. Here we show that 2-AG levels are inversely associated with fear generalization at the behavioral level in both mice and humans. In mice, 2-AG depletion increases the proportion of neurons, and the similarity between neuronal representations, of threat-predictive and neutral stimuli within prelimbic prefrontal cortex ensembles. In humans, increased dorsolateral prefrontal cortical-amygdala resting state connectivity is inversely correlated with fear generalization. These data provide convergent cross-species evidence that 2-AG is a key regulator of fear generalization and suggest 2-AG deficiency could represent a trauma-related disorder susceptibility endophenotype.

BNST GluN2D-containing NMDARs contribute to ethanol intake but not negative affective behaviors in female mice.

BACKGROUND: Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST) and Crh+ neurons in this region play a key role in chronic ethanol-induced increases in volitional intake, hypothesized to be driven by emergent negative affective behaviors. Excitatory N-methyl-d-aspartate receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. Specifically, GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. METHODS: Male and female mice underwent a home cage chronic drinking forced abstinence model (CDFA) to assess the impact of 1 day or 2 weeks of ethanol abstinence on BNST synaptic transmission and BNST Grin gene expression. Constitutive and conditional BNST GluN2D knockout mice were used to assess the impact of GluN2D deletion on continuous access ethanol intake as well as negative affect behaviors, using the open field, elevated zero maze, and forced swim tasks. RESULTS: We report here that excitatory transmission undergoes time-dependent upregulation in BNST Crh+ cells. Further, knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in the dBNST or dBNST Crh+ neurons. Finally, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. CONCLUSIONS: These data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking but not ethanol abstinence, highlighting sex differences and behavioral specificity. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

BNST GluN2D-containing NMDARs contribute to ethanol intake but not negative affective behaviors in female mice.

Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), and Crh + neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNST Crh + cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in dBNST or specifically in dBNST Crh + neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

Blood-brain barrier remodeling in an organ-on-a-chip device shows Dkk1 to be a regulator of early metastasis.

Brain metastases are the most lethal progression event, in part because the biological processes underpinning brain metastases are poorly understood. There is a paucity of realistic models of metastasis, as current in vivo murine models are slow to manifest metastasis. We set out to delineate metabolic and secretory modulators of brain metastases by utilizing two models consisting of in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the blood-brain-barrier and niche; and 2) a migration chip that assesses cell migration. We report secretory cues provided by the brain niche that attract metastatic cancer cells to colonize the brain niche region. Astrocytic Dkk-1 is increased in response to brain-seeking breast cancer cells and stimulates cancer cell migration. Brain-metastatic cancer cells under Dkk-1 stimulation increase gene expression of FGF-13 and PLCB1. Further, extracellular Dkk-1 modulates cancer cell migration upon entering the brain niche.

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.

Endocannabinoid Signaling Collapse Mediates Stress-Induced Amygdalo-Cortical Strengthening.

Functional coupling between the amygdala and the dorsomedial prefrontal cortex (dmPFC) has been implicated in the generation of negative affective states; however, the mechanisms by which stress increases amygdala-dmPFC synaptic strength and generates anxiety-like behaviors are not well understood. Here, we show that the mouse basolateral amygdala (BLA)-prelimbic prefrontal cortex (plPFC) circuit is engaged by stress and activation of this pathway in anxiogenic. Furthermore, we demonstrate that acute stress exposure leads to a lasting increase in synaptic strength within a reciprocal BLA-plPFC-BLA subcircuit. Importantly, we identify 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling as a key mechanism limiting glutamate release at BLA-plPFC synapses and the functional collapse of multimodal 2-AG signaling as a molecular mechanism leading to persistent circuit-specific synaptic strengthening and anxiety-like behaviors after stress exposure. These data suggest that circuit-specific impairment in 2-AG signaling could facilitate functional coupling between the BLA and plPFC and the translation of environmental stress to affective pathology.

An endocannabinoid-regulated basolateral amygdala-nucleus accumbens circuit modulates sociability.

Deficits in social interaction (SI) are a core symptom of autism spectrum disorders (ASDs); however, treatments for social deficits are notably lacking. Elucidating brain circuits and neuromodulatory signaling systems that regulate sociability could facilitate a deeper understanding of ASD pathophysiology and reveal novel treatments for ASDs. Here we found that in vivo optogenetic activation of the basolateral amygdala-nucleus accumbens (BLA-NAc) glutamatergic circuit reduced SI and increased social avoidance in mice. Furthermore, we found that 2-arachidonoylglycerol (2-AG) endocannabinoid signaling reduced BLA-NAc glutamatergic activity and that pharmacological 2-AG augmentation via administration of JZL184, a monoacylglycerol lipase inhibitor, blocked SI deficits associated with in vivo BLA-NAc stimulation. Additionally, optogenetic inhibition of the BLA-NAc circuit markedly increased SI in the Shank3B-/- mouse, an ASD model with substantial SI impairment, without affecting SI in WT mice. Finally, we demonstrated that JZL184 delivered systemically or directly to the NAc also normalized SI deficits in Shank3B-/- mice, while ex vivo JZL184 application corrected aberrant NAc excitatory and inhibitory neurotransmission and reduced BLA-NAc-elicited feed-forward inhibition of NAc neurons in Shank3B-/- mice. These data reveal circuit-level and neuromodulatory mechanisms regulating social function relevant to ASDs and suggest 2-AG augmentation could reduce social deficits via modulation of excitatory and inhibitory neurotransmission in the NAc.

Breast cancers utilize hypoxic glycogen stores via PYGB, the brain isoform of glycogen phosphorylase, to promote metastatic phenotypes.

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer.

A platform for artificial intelligence based identification of the extravasation potential of cancer cells into the brain metastatic niche.

Brain metastases are the most lethal complication of advanced cancer; therefore, it is critical to identify when a tumor has the potential to metastasize to the brain. There are currently no interventions that shed light on the potential of primary tumors to metastasize to the brain. We constructed and tested a platform to quantitatively profile the dynamic phenotypes of cancer cells from aggressive triple negative breast cancer cell lines and patient derived xenografts (PDXs), generated from a primary tumor and brain metastases from tumors of diverse organs of origin. Combining an advanced live cell imaging algorithm and artificial intelligence, we profile cancer cell extravasation within a microfluidic blood-brain niche (µBBN) chip, to detect the minute differences between cells with brain metastatic potential and those without with a PPV of 0.91 in the context of this study. The results show remarkably sharp and reproducible distinction between cells that do and those which do not metastasize inside of the device.

Detection of Cyclooxygenase-2-Derived Oxygenation Products of the Endogenous Cannabinoid 2-Arachidonoylglycerol in Mouse Brain.

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX-2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC's extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC's hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM "primes" the IBC cells' cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins -6, -8, and -10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Presymptomatic glutamate levels in prefrontal cortex in the Hdh(CAG150) mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a genetic neurodegenerative disorder with few available treatments. Clinical observations suggest prefrontal dysfunction in early stages of HD is associated with altered glutamate transport. Evidence from the R6/2 mouse model suggests an abnormal increase in glutamate signaling in the sensorimotor cortex and striatum. OBJECTIVE: The present study was designed to determine if a similar deficit in glutamate function occurs in the prefrontal cortex (PFC) of Hdh(CAG150) mice. METHODS: We used the following groups of 40 week old male and female Hdh(CAG150) mice: homozygote n = 7, heterozygote n = 7, wild type n = 6. Motor coordination was evaluated using a hanging wire grid test and a balance beam. Microdialysis measurements were taken from the PFC of freely moving mice while glutamate transporters were inhibited by L-trans-pyrrolidine-2, 4-dicarboxylate (PDC) and compared to baseline glutamate levels. RESULTS: RESULTS indicated an elevation in glutamate levels in response to PDC but no significant difference among genotype groups. When comparing wild type and homozygote alone, a significant difference in total extracellular glutamate was observed. Contrary to our original hypothesis, the homozygote group had lower glutamate levels compared to their wild type counterparts. Furthermore, there was a significant difference in GABA measurements across genotypes. CONCLUSIONS: Our results suggest a mechanistic dichotomy between R6/2 and Hdh(CAG150) mice and underscores the need to select the appropriate HD mouse model when assessing therapeutic interventions. In particular, the time when animals are evaluated can have a significant impact on behavioral and physiological measures and so should be carefully considered.