A covalent small molecule inhibitor of glutamate-oxaloacetate transaminase 1 impairs pancreatic cancer growth.

Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA. However, the development of GOT1 inhibitors has been challenging, and no compound has yet demonstrated selectivity for GOT1-dependent cell metabolism or selective growth inhibition of PDA cell lines. In contrast, potent inhibitors that covalently bind to the transaminase cofactor pyridoxal-5'-phosphate (PLP), within the active site of the enzyme, have been reported for kynurenine aminotransferase (KAT) and gamma-aminobutyric acid aminotransferase (GABA-AT). Given the drug discovery successes with these transaminases, we aimed to identify PLP-dependent suicide substrate-type GOT1 inhibitors. Here, we demonstrate that PF-04859989, a known KAT2 inhibitor, has PLP-dependent inhibitory activity against GOT1 and shows selective growth inhibition of PDA cell lines.

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus.

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine-seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath-application of NA induced similar levels of hyperpolarization in both saline- and cocaine-treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine- but not saline-treated neurons through α2 adrenoceptors. This NA-induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired-pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.

The role of neuroplasticity in cholinergic neurons of the laterodorsal tegmental nucleus for cocaine addiction.

A large body of literature indicates that neural adaptations induced by cocaine in the mesocorticolibic system cause addictive behaviors. Emerging evidence suggests that the laterdorsal tegmental nucleus (LDT), which contains cholinergic, glutamatergic and GABAergic neurons and innervates the ventral tegmental area (VTA), might also contribute to the development of cocaine addiction. In this review, we summarize our recent findings showing that neuroplasticity elicited by cocaine administration in LDT cholinergic neurons is involved in the expression of addictive behaviors. Ex vivo electrophysiological recordings obtained from repeatedly cocaine administered rats revealed and increased excitatory synaptic transmission to and enhanced intrinsic membrane excitability in LDT cholinergic neurons. The former depended on enhanced glutamate release probability form presynaptic terminals and the latter was mediated by increased persistent sodium conductance. Additionally, intra-LDT administration of AMPA/HMDA receptor antagonists or a persistent sodium channel blocker attenuated the expression of cocaine-induced conditioned place preference. These findings suggest that chronic cocaine exposure-induced neuroplasticity in LDT cholinergic neurons may activate LDT cholinergic neurons, which in turn may enhance the activity of dopamine neurons in the VTA, leading to the development of cocaine addiction.

Intrinsic membrane plasticity via increased persistent sodium conductance of cholinergic neurons in the rat laterodorsal tegmental nucleus contributes to cocaine-induced addictive behavior.

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus implicated in reward processing and is one of the main sources of cholinergic afferents to the ventral tegmental area (VTA). Neuroplasticity in this structure may affect the excitability of VTA dopamine neurons and mesocorticolimbic circuitry. Here, we provide evidence that cocaine-induced intrinsic membrane plasticity in LDT cholinergic neurons is involved in addictive behaviors. After repeated experimenter-delivered cocaine exposure, ex vivo whole-cell recordings obtained from LDT cholinergic neurons revealed an induction of intrinsic membrane plasticity in regular- but not burst-type neurons, resulting in increased firing activity. Pharmacological examinations showed that increased riluzole-sensitive persistent sodium currents, but not changes in Ca(2+) -activated BK, SK or voltage-dependent A-type potassium conductance, mediated this plasticity. In addition, bilateral microinjection of riluzole into the LDT immediately before the test session in a cocaine-induced conditioned place preference (CPP) paradigm inhibited the expression of cocaine-induced CPP. These findings suggest that intrinsic membrane plasticity in LDT cholinergic neurons is causally involved in the development of cocaine-induced addictive behaviors.

[Involvement and plasticity of brainstem cholinergic neurons in cocaine-induced addiction].

Although the involvement and plasticity of the mesocorticolimbic dopamine (DA) system in cocaine-induced addiction have been studied extensively, the role of the brainstem cholinergic system in cocaine addiction remains largely unexplored. The laterodorsal tegmental nucleus (LDT) contains cholinergic neurons that innervate the ventral tegmental area (VTA) and is crucial for regulating the activity of VTA DA neurons, implying that LDT may also be associated with cocaine addiction. In this review, we summarize our recent findings showing that cholinergic transmission from the LDT to the VTA is involved in acquisition and expression of cocaine-induced conditioned place preference and that, after repeated cocaine exposures, these neurons exhibit synaptic plasticity, which is dependent on NMDA receptor activation, nitric oxide production, and the activity of medial prefrontal cortex. The findings strongly suggest that LDT cholinergic neurons may critically contribute to developing cocaine-induced addiction.

Cocaine exposure enhances excitatory synaptic drive to cholinergic neurons in the laterodorsal tegmental nucleus.

Accumulating evidence indicates that the laterodorsal tegmental nucleus (LDT) is associated with reward processing and addiction. The cholinergic projection from the LDT to the ventral tegmental area is essential for a large dopamine release in the nucleus accumbens, which is critically involved in the reinforcing effects of addictive drugs, including cocaine. In contrast to the large number of studies on plasticity induced after cocaine exposure in the mesocorticolimbic dopaminergic system, it remains unknown whether LDT cholinergic neurons exhibit plastic changes following cocaine administration. To address this issue, we performed ex vivo whole-cell recordings in LDT cholinergic neurons obtained from rats following cocaine administration. Neurons obtained from 1 day after 5-day cocaine-treated rats showed significantly smaller paired-pulse ratios of evoked EPSCs and higher miniature EPSC frequencies than those from saline-treated rats, indicating an induction of presynaptic plasticity of increased glutamate release. This plasticity seemed to recover after a 5-day withdrawal from repeated cocaine exposure, and required NMDA receptor stimulation and nitric oxide production. Additionally, pharmacological suppression of activity of the medial prefrontal cortex inhibited the presynaptic plasticity in the LDT. On the other hand, AMPA/NMDA ratios were not different between saline- and cocaine-treated groups, revealing an absence of postsynaptic plasticity. These findings provide the first direct evidence of cocaine-induced synaptic plasticity in LDT cholinergic neurons and suggest that the presynaptic plasticity enhances the activity of LDT cholinergic neurons, contributing to the expression of cocaine-induced addictive behaviors through the dysregulation of the mesocorticolimbic system.

Potentiality of multiple modalities for single-cell analyses to evaluate the tumor microenvironment in clinical specimens.

Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

Nitric oxide in the medial prefrontal cortex contributes to the acquisition of cocaine place preference and synaptic plasticity in the laterodorsal tegmental nucleus.

Nitric oxide (NO), a gaseous neurotransmitter, is involved in a variety of brain functions, including drug addiction. Although previous studies have suggested that NO plays an important role in the development of cocaine addiction, the brain region(s) in which NO acts and how it contributes to cocaine addiction remain unclear. In this study, we examined these issues using a cocaine-induced conditioned place preference (CPP) paradigm and ex vivo electrophysiological recordings in rats. Specifically, we focused on the medial prefrontal cortex (mPFC) and laterodorsal tegmental nucleus (LDT), brain regions associated with cocaine CPP development and cocaine-induced plasticity. Intra-mPFC injection of the non-selective NO synthase (NOS) inhibitor L-NAME or the neuronal NOS (nNOS) selective inhibitor L-NPA during the conditioning phase disrupted cocaine CPP. Additionally, intra-mPFC injection of L-NPA prior to each cocaine injection prevented the induction of presynaptic plasticity, induced by repeated cocaine administration, in LDT cholinergic neurons. These findings indicate that NO generated in the mPFC contributes to the acquisition of cocaine CPP and the induction of neuroplasticity in LDT cholinergic neurons. Together with previous studies showing that NO induces membrane plasticity in mPFC neurons, that mPFC neurons project to the LDT, and that LDT activity is critical for the acquisition of cocaine CPP, the present findings suggest that NO-mediated neuroplasticity induced in the mPFC-LDT circuitry is critical for the development of cocaine addiction.