Persistent enhancement of basolateral amygdala-dorsomedial striatum synapses causes compulsive-like behaviors in mice.

Compulsive behaviors are observed in a range of psychiatric disorders, however the neural substrates underlying the behaviors are not clearly defined. Here we show that the basolateral amygdala-dorsomedial striatum (BLA-DMS) circuit activation leads to the manifestation of compulsive-like behaviors. We revealed that the BLA neurons projecting to the DMS, mainly onto dopamine D1 receptor-expressing neurons, largely overlap with the neuronal population that responds to aversive predator stress, a widely used anxiogenic stressor. Specific optogenetic activation of the BLA-DMS circuit induced a strong anxiety response followed by compulsive grooming. Furthermore, we developed a mouse model for compulsivity displaying a wide spectrum of compulsive-like behaviors by chronically activating the BLA-DMS circuit. In these mice, persistent molecular changes at the BLA-DMS synapses observed were causally related to the compulsive-like phenotypes. Together, our study demonstrates the involvement of the BLA-DMS circuit in the emergence of enduring compulsive-like behaviors via its persistent synaptic changes.

PLCγ1 in dopamine neurons critically regulates striatal dopamine release via VMAT2 and synapsin III.

Dopamine neurons are essential for voluntary movement, reward learning, and motivation, and their dysfunction is closely linked to various psychological and neurodegenerative diseases. Hence, understanding the detailed signaling mechanisms that functionally modulate dopamine neurons is crucial for the development of better therapeutic strategies against dopamine-related disorders. Phospholipase Cγ1 (PLCγ1) is a key enzyme in intracellular signaling that regulates diverse neuronal functions in the brain. It was proposed that PLCγ1 is implicated in the development of dopaminergic neurons, while the physiological function of PLCγ1 remains to be determined. In this study, we investigated the physiological role of PLCγ1, one of the key effector enzymes in intracellular signaling, in regulating dopaminergic function in vivo. We found that cell type-specific deletion of PLCγ1 does not adversely affect the development and cellular morphology of midbrain dopamine neurons but does facilitate dopamine release from dopaminergic axon terminals in the striatum. The enhancement of dopamine release was accompanied by increased colocalization of vesicular monoamine transporter 2 (VMAT2) at dopaminergic axon terminals. Notably, dopamine neuron-specific knockout of PLCγ1 also led to heightened expression and colocalization of synapsin III, which controls the trafficking of synaptic vesicles. Furthermore, the knockdown of VMAT2 and synapsin III in dopamine neurons resulted in a significant attenuation of dopamine release, while this attenuation was less severe in PLCγ1 cKO mice. Our findings suggest that PLCγ1 in dopamine neurons could critically modulate dopamine release at axon terminals by directly or indirectly interacting with synaptic machinery, including VMAT2 and synapsin III.

GABAergic-like dopamine synapses in the brain.

Dopamine synapses play a crucial role in volitional movement and reward-related behaviors, while dysfunction of dopamine synapses causes various psychiatric and neurological disorders. Despite this significance, the true biological nature of dopamine synapses remains poorly understood. Here, we show that dopamine transmission is strongly correlated with GABA co-transmission across the brain and dopamine synapses are structured and function like GABAergic synapses with marked regional heterogeneity. In addition, GABAergic-like dopamine synapses are clustered on the dendrites, and GABA transmission at dopamine synapses has distinct physiological properties. Interestingly, the knockdown of neuroligin-2, a key postsynaptic protein at GABAergic synapses, unexpectedly does not weaken GABA co-transmission but instead facilitates it at dopamine synapses in the striatal neurons. More importantly, the attenuation of GABA co-transmission precedes deficits in dopaminergic transmission in animal models of Parkinson's disease. Our findings reveal the spatial and functional nature of GABAergic-like dopamine synapses in health and disease.

Deficiency in RCAT-1 Function Causes Dopamine Metabolism Related Behavioral Disorders in Caenorhabditis elegans.

When animals are faced with food depletion, food search-associated locomotion is crucial for their survival. Although food search-associated locomotion is known to be regulated by dopamine, it has yet to investigate the potential molecular mechanisms governing the regulation of genes involved in dopamine metabolism (e.g., cat-1, cat-2) and related behavioral disorders. During the studies of the pheromone ascaroside, a signal of starvation stress in C. elegans, we identified R02D3.7, renamed rcat-1 (regulator of cat genes-1), which had previously been shown to bind to regulatory sequences of both cat-1 and cat-2 genes. It was found that RCAT-1 (R02D3.7) is expressed in dopaminergic neurons and functions as a novel negative transcriptional regulator for cat-1 and cat-2 genes. When a food source becomes depleted, the null mutant, rcat-1(ok1745), exhibited an increased frequency of high-angled turns and intensified area restricted search behavior compared to the wild-type animals. Moreover, rcat-1(ok1745) also showed defects in state-dependent olfactory adaptation and basal slowing response, suggesting that the mutants are deficient in either sensing food or locomotion toward food. However, rcat-1(ok1745) has normal cuticular structures and locomotion genes. The discovery of rcat-1 not only identifies a new subtype of dopamine-related behaviors but also provides a potential therapeutic target in Parkinson's disease.

Dopaminergic Control of the Feeding Circuit.

There is increasing evidence demonstrating that reward-related motivational food intake is closely connected with the brain's homeostatic system of energy balance and that this interaction might be important in the integrative control of feeding behavior. Dopamine regulates motivational behavior, including feeding behaviors, and the dopamine reward system is recognized as the most prominent system that controls appetite and motivational and emotional drives for food. It appears that the dopamine system exerts a critical role in the control of feeding behavior not only by the reward-related circuit, but also by contributing to the homeostatic circuit of food intake, suggesting that dopamine plays an integrative role across the converging circuitry of control of food intake by linking energy state-associated signals to reward-related behaviors. This review will cover and discuss up-to-date findings on the dopaminergic control of food intake by both the reward-related circuit and the homeostatic hypothalamic system.

Stress and the dopaminergic reward system.

Dopamine regulates reward-related behavior through the mesolimbic dopaminergic pathway. Stress affects dopamine levels and dopaminergic neuronal activity in the mesolimbic dopamine system. Changes in mesolimbic dopaminergic neurotransmission are important for coping with stress, as they allow adaption to behavioral responses to various environmental stimuli. Upon stress exposure, modulation of the dopaminergic reward system is necessary for monitoring and selecting the optimal process for coping with stressful situations. Aversive stressful events may negatively regulate the dopaminergic reward system, perturbing reward sensitivity, which is closely associated with chronic stress-induced depression. The mesolimbic dopamine system is excited not only by reward but also by aversive stressful stimuli, which adds further intriguing complexity to the relationship between stress and the reward system. This review focuses on lines of evidence related to how stress, especially chronic stress, affects the mesolimbic dopamine system, and discusses the role of the dopaminergic reward system in chronic stress-induced depression.

Potential Regulatory Role of Human-Carboxylesterase-1 Glycosylation in Liver Cancer Cell Growth.

We previously reported that human carboxylesterase 1 (CES1), a serine esterase containing a unique N-linked glycosyl group at Asn79 (N79 CES1), is a candidate serological marker of hepatocellular carcinoma (HCC). CES1 is normally present at low-to-undetectable levels in normal human plasma, HCC tumors, and major liver cancer cell lines. To investigate the potential mechanism underlying the suppression of CES1 expression in liver cancer cells, we took advantage of the low detectability of this marker in tumors by overexpressing CES1 in multiple HCC cell lines, including stable Hep3B cells. We found that the population of CES1-overexpressing (OE) cells decreased and that their doubling time was longer compared with mock control liver cancer cells. Using interactive transcriptome, proteome, and subsequent Gene Ontology enrichment analysis of CES1-OE cells, we found substantial decreases in the expression levels of genes involved in cell cycle regulation and proliferation. This antiproliferative function of the N79 glycan of CES1 was further supported by quantitative real-time polymerase chain reaction, flow cytometry, and an apoptosis protein array assay. An analysis of the levels of key signaling target proteins via Western blotting suggested that CES1 overexpression exerted an antiproliferative effect via the PKD1/PKCµ signaling pathway. Similar results were also seen in another HCC cell line (PLC/RFP/5) after transient transfection with CES1 but not in similarly treated non-HCC cell lines (e.g., HeLa and Tera-1 cells), suggesting that CES1 likely exerts a liver cell-type-specific suppressive effect. Given that the N-linked glycosyl group at Asn79 (N79 glycan) of CES1 is known to influence CES1 enzyme activity, we hypothesized that the post-translational modification of CES1 at N79 may be linked to its antiproliferative activity. To investigate the regulatory effect of the N79 glycan on cellular growth, we mutated the single N-glycosylation site in CES1 from Asn to Gln (CES1-N79Q) via site-directed mutagenesis. Fluorescence 2-D difference gel electrophoresis protein expression analysis of cell lysates revealed an increase in cell growth and a decrease in doubling time in cells carrying the N79Q mutation. Thus our results suggest that CES1 exerts an antiproliferative effect in liver cancer cells and that the single N-linked glycosylation at Asn79 plays a potential regulatory role. These functions may underlie the undetectability of CES1 in human HCC tumors and liver cancer cell lines. Mass spectrometry data are available via ProteomeXchange under the identifier PXD021573.

Dynamic Changes in the Bridging Collaterals of the Basal Ganglia Circuitry Control Stress-Related Behaviors in Mice.

The basal ganglia network has been implicated in the control of adaptive behavior, possibly by integrating motor learning and motivational processes. Both positive and negative reinforcement appear to shape our behavioral adaptation by modulating the function of the basal ganglia. Here, we examined a transgenic mouse line (G2CT) in which synaptic transmissions onto the medium spiny neurons (MSNs) of the basal ganglia are depressed. We found that the level of collaterals from direct pathway MSNs in the external segment of the globus pallidus (GPe) ('bridging collaterals') was decreased in these mice, and this was accompanied by behavioral inhibition under stress. Furthermore, additional manipulations that could further decrease or restore the level of the bridging collaterals resulted in an increase in behavioral inhibition or active behavior in the G2CT mice, respectively. Collectively, our data indicate that the striatum of the basal ganglia network integrates negative emotions and controls appropriate coping responses in which the bridging collateral connections in the GPe play a critical regulatory role.

The function of PLCγ1 in developing mouse mDA system.

During neural development, growing neuronal cells consistently sense and communicate with their surroundings through the use of signaling molecules. In this process, spatiotemporally well-coordinated intracellular signaling is a prerequisite for proper neuronal network formation. Thus, intense interest has focused on investigating the signaling mechanisms in neuronal structure formation that link the activation of receptors to the control of cell shape and motility. Recent studies suggest that Phospholipase C gamma1 (PLCγ1), a signal transducer, plays key roles in nervous system development by mediating specific ligand-receptor systems. In this overview of the most recent advances in the field, we discuss the mechanisms by which extracellular stimuli trigger PLCγ1 signaling and, the role PLCγ1 in nervous system development.

MiR-195 and miR-497 suppress tumorigenesis in lung cancer by inhibiting SMURF2-induced TGF-ß receptor I ubiquitination.

SMURF2 is a member of the HECT family of E3 ubiquitin ligases that have important roles as a negative regulator of transforming growth factor-ß (TGF-ß) signaling through ubiquitin-mediated degradation of TGF-ß receptor I. However, the regulatory mechanism of SMURF2 is largely unknown. In this study, we identified that micro(mi)R-195 and miR-497 putatively target SMURF2 using several target prediction databases. Both miR-195 and miR-497 bind to the 3'-UTR of the SMURF2 mRNA and inhibit SMURF2 expression. Furthermore, miR-195 and miR-497 regulate SMURF2-dependent TßRI ubiquitination and cause the activation of the TGF-ß signaling pathway in lung cancer cells. Upregulation of miR-195 and miR-497 significantly reduced cell viability and colony formation through the activation of TGF-ß signaling. Interestingly, miR-195 and miR-497 also reduced the invasion ability of lung cancer cells when cells were treated with TGF-ß1. Subsequent in vivo studies in xenograft nude mice model revealed that miR-195 and miR-497 repress tumor growth. These findings demonstrate that miR-195 and miR-497 act as a tumor suppressor by suppressing ubiquitination-mediated degradation of TGF-ß receptors through SMURF2, and suggest that miR-195 and miR-497 are potential therapeutic targets for lung cancer.

Pan-HDAC Inhibitors Promote Tau Aggregation by Increasing the Level of Acetylated Tau.

Epigenetic remodeling via histone acetylation has become a popular therapeutic strategy to treat Alzheimer's disease (AD). In particular, histone deacetylase (HDAC) inhibitors including M344 and SAHA have been elucidated to be new drug candidates for AD, improving cognitive abilities impaired in AD mouse models. Although emerged as a promising target for AD, most of the HDAC inhibitors are poorly selective and could cause unwanted side effects. Here we show that tau is one of the cytosolic substrates of HDAC and the treatment of HDAC inhibitors such as Scriptaid, M344, BML281, and SAHA could increase the level of acetylated tau, resulting in the activation of tau pathology.

Dopamine D2 receptor-mediated circuit from the central amygdala to the bed nucleus of the stria terminalis regulates impulsive behavior.

Impulsivity is closely associated with addictive disorders, and changes in the brain dopamine system have been proposed to affect impulse control in reward-related behaviors. However, the central neural pathways through which the dopamine system controls impulsive behavior are still unclear. We found that the absence of the D2 dopamine receptor (D2R) increased impulsive behavior in mice, whereas restoration of D2R expression specifically in the central amygdala (CeA) of D2R knockout mice (Drd2-/-) normalized their enhanced impulsivity. Inhibitory synaptic output from D2R-expressing neurons in the CeA underlies modulation of impulsive behavior because optogenetic activation of D2R-positive inhibitory neurons that project from the CeA to the bed nucleus of the stria terminalis (BNST) attenuate such behavior. Our identification of the key contribution of D2R-expressing neurons in the CeA → BNST circuit to the control of impulsive behavior reveals a pathway that could serve as a target for approaches to the management of neuropsychiatric disorders associated with impulsivity.

Glucose-regulated protein 78 binds to and regulates the melanocortin-4 receptor.

The melanocortin-4 receptor (MC4R) belongs to the G protein-coupled receptor (GPCR) family and plays an essential role in the control of energy homeostasis. Here, we identified a novel MC4R-interacting protein, glucose-regulated protein 78 (GRP78), from a pulldown assay using hypothalamic protein extracts and the third intracellular loop of MC4R. We found that MC4R interacted with GRP78 in both the cytosol and at the cell surface and that this interaction increased when MC4R was internalized in the presence of the agonist melanotan-II (MTII). Downregulation of GRP78 using a short interfering RNA approach attenuated MTII-mediated receptor internalization. Reduction in GRP78 expression during tunicamycin-induced endoplasmic reticulum stress also suppressed MTII-mediated internalization of MC4R and cAMP-mediated transcriptional activity. Furthermore, lentiviral-mediated short hairpin RNA knockdown of endogenous GRP78 in the paraventricular nucleus (PVN) of the hypothalamus resulted in an increase in body weight in mice fed a high-fat diet. These results suggest that GRP78 in the PVN binds to MC4R and may have a chaperone-like role in the regulation of MC4R trafficking and signaling.

Nootropic nanocomplex with enhanced blood-brain barrier permeability for treatment of traumatic brain injury-associated neurodegeneration.

Traumatic brain injury (TBI) is an intracranial injury which can induce immediate neuroinflammation and long-term neurological deficits. Methylene blue (MB) as a nootropic has a great potential to treat neurodegeneration after TBI because of its anti-inflmmatory and neuroprotective functions. However, its limited accumulation to the brain across the blood-brain barrier (BBB) remains a major hurdle to be overcome. In this paper, we present a polymer surfactant-encapsulated nanocomplex of MB as a delivery system with high BBB permeability for efficacious treatment of TBI-induced neurodegeneration. MB was formulated via electrostatically/hydrophobically directed assembly with fatty acid and Pluronic surfactant (F-127 or F-68) to construct nanocomplexes of two different colloidal sizes (<10 nm and ~108 nm in hydrodynamic diameter for NanoMB-127 and NanoMB-68, respectively). Compared to uncomplexed free MB, formulation into the ultrasmall nanocomplex (NanoMB-127) significantly enhanced the uptake of MB by blood-brain vascular endothelial bEnd3 cells in vitro, and indeed improved its BBB penetration upon systemic administration to normal mice in vivo. However, large-size NanoMB-68 showed negligible BBB crossing despite the efficient bEnd3 cell internalization in vitro, probably due to the unfavorable pharmacokinetic profile associated with its large particle size. By virtue of the efficient BBB penetration and cellular uptake, ultrasmall NanoMB-127 was shown to distinctively reduce the expression level of an inflammatory cytokine with no notable toxicity in vitro and also considerably prevent the neurodegeneration after TBI in mice at much lower doses than free MB. Overall, the Pluronic-supported nanocomplexation method allows efficient brain delivery of MB, offering a novel way of enhancing the efficacy of neurotherapeutics to treat brain diseases.

Hypomyelination and cognitive impairment in mice lacking CD133 (Prominin-1).

The CD133 antigen, also known as prominin-1, is a glycoprotein that specifically localizes to plasma membrane protrusions. The precise function of CD133 remains unknown, but it is expressed in various progenitor cells including those derived from the neural and hematopoietic system, as well as different tissues. In the adult mouse brain, CD133 is highly expressed in white matter. Here, we performed immunohistochemical staining and electron microscopy to demonstrate that mice lacking CD133 (CD133-/-) exhibit decreased myelin in the corpus callosum, the largest white matter tract in the brain. Hypomyelination in CD133-/- mice was associated with fewer oligodendrocyte progenitor cells and mature oligodendrocytes. Behavioral analyses revealed that significantly impaired object recognition memory and altered Y-maze performance by CD133-/- mice compared with wild-type mice, suggesting perturbed cognitive performance. These results suggest that CD133 regulates myelination and understanding the underlying molecular mechanisms may guide the development of novel therapeutic strategies for diseases characterized by myelin deficiency.

Publisher Correction: Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss.

Dopaminergic systems play a major role in reward-related behavior and dysregulation of dopamine (DA) systems can cause several mental disorders, including depression. We previously reported that dopamine D2 receptor knockout (D2R-/-) mice display increased anxiety and depression-like behaviors upon chronic stress. Here, we observed that chronic stress caused myelin loss in wild-type (WT) mice, while the myelin level in D2R-/- mice, which was already lower than that in WT mice, was not affected upon stress. Fewer mature oligodendrocytes (OLs) were observed in the corpus callosum of stressed WT mice, while in D2R-/- mice, both the control and stressed group displayed a decrease in the number of mature OLs. We observed a decrease in the number of active ß-catenin (ABC)-expressing and TCF4-expressing cells among OL lineage cells in the corpus callosum of stressed WT mice, while such regulation was not found in D2R-/- mice. Administration of lithium normalized the behavioral impairments and myelin damage induced by chronic stress in WT mice, and restored the number of ABC-positive and TCF4-positive OLs, while such effect was not found in D2R-/- mice. Together, our findings indicate that chronic stress induces myelin loss through the Wnt/ß-catenin signaling pathway in association with DA signaling through D2R.

Effect of optogenetic manipulation of accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization.

Repetitive exposure to addictive drugs causes synaptic modification in the mesocorticolimbic dopamine (DA) system. Dopamine D1 receptors (D1R) or D2 receptors (D2R) expressed in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc) play critical roles in the control of addictive behaviors. Optogenetic activation of D2R-expressing MSNs (D2R-MSNs) in the NAc previously demonstrated that these neurons play a key role in withdrawal-induced plasticity. Here, we examined the effect of optogenetic inhibition of D2R-MSNs in the NAc on cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding archaerhodopsin (ArchT) were delivered into the NAc of D2-Cre transgenic mice. Activation of ArchT produced photoinhibition of D2R-MSNs and caused disinhibition of neighboring MSNs in the NAc. However, such optogenetic silencing of D2R-MSNs in the NAc in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. Similarly, photoinhibition of NAc D2R-MSNs in the NAc during the drug withdrawal period did not affect the expression of cocaine-induced behavioral sensitization. More detailed analysis of the effects of optogenetic activation of D2R-MSNs suggests that D2R-MSNs in the NAc exert important modulatory effects on neighboring MSN neurons, which may control the balanced output of NAc MSNs to control addictive behaviors.

Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex.

Misfolded polypeptides are rapidly cleared from cells via the ubiquitin-proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF-eEF1A1-DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.

MIR-27a regulates the TGF-ß signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

The transforming growth factor-ß (TGF-ß) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-ß signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-ß-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.