Hallucinogens, including mescaline, psilocybin, and lysergic acid diethylamide (LSD), profoundly affect perception, cognition, and mood. All known drugs of this class are 5-HT(2A) receptor (2AR) agonists, yet closely related 2AR agonists such as lisuride lack comparable psychoactive properties. Why only certain 2AR agonists are hallucinogens and which neural circuits mediate their effects are poorly understood. By genetically expressing 2AR only in cortex, we show that 2AR-regulated pathways on cortical neurons are sufficient to mediate the signaling pattern and behavioral response to hallucinogens. Hallucinogenic and nonhallucinogenic 2AR agonists both regulate signaling in the same 2AR-expressing cortical neurons. However, the signaling and behavioral responses to the hallucinogens are distinct. While lisuride and LSD both act at 2AR expressed by cortex neurons to regulate phospholipase C, LSD responses also involve pertussis toxin-sensitive heterotrimeric G(i/o) proteins and Src. These studies identify the long-elusive neural and signaling mechanisms responsible for the unique effects of hallucinogens.

The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception 1,2 . Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT 2A receptors (2AR) 3,4 . Drugs that interact with metabotropic glutamate receptors (mGluR) also show potential for the treatment of schizophrenia [5][6][7] . The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide (LSD), require the 2AR [8][9][10] and resemble some of the core symptoms of schizophrenia [10][11][12] . Here we show that the mGluR2 interacts via specific transmembrane helix domains with the 2AR, a member of an unrelated G protein-coupled receptor (GPCR) family, to form functional complexes in brain cortex. The 2AR/mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen specific signalling and behavioural responses. In postmortem human brain from untreated schizophrenic subjects, the 2AR is up-regulated and the mGluR2 is down-regulated, a pattern that could predispose to psychosis. These regulatory changes suggest that the 2AR/mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and represents a promising new target for the treatment of psychosis.Correspondence and requests for materials should be addressed to: J.G.M (e-mail: Javier.Maeso@mssm.edu) S.C.S. (e-mail: Stuart.Sealfon@mssm.edu). The 2AR and mGluR2/3 show an overlapping distribution in brain cortex in autoradiography studies 13 . The mGluR2 and mGluR3 are not distinguished by autoradiographic ligands. We used fluorescent in situ hybridization (FISH) to determine whether either of these receptor subtypes are co-expressed by the same neurons. In layer V mouse somatosensory cortex (SCx), 2AR mRNA positive cells were mostly mGluR2 mRNA positive. The level of expression in SCx was much lower for mGluR3 mRNA, which rarely co-localized with 2AR mRNA (Fig. 1a). Control studies validated assay sensitivity and specificity, and similar 2AR/mGluR2 mRNA co-localization was found in cortical primary cultures (Figs. 1a,b,c, and Supplementary Fig. S1). Translation of 2AR protein in cortical pyramidal neurons was found to be necessary for normal mGluR2 expression. Mice with globally disrupted 2AR expression (htr2A−/− mice) showed reduced cortical mGluR2 binding and expression, while mice in which 2AR expression was selectively restored in cortical pyramidal neurons 8,14 showed control expression levels (Supplementary Table S1, and Supplementary Fig. S2). The effects of mGluR2/3 activation on 2AR responses have been generally attributed to synaptic mechanisms 5,6,13,15 . However, the co-localization of 2AR and mGluR2 and the reduction of mGluR2 expression levels in htr2A−/− mice motivated us to examine whether a direct mechanism contributed to cortical crosstalk between these two receptor systems. NIH Public AccessRecent studies have demonstrated that some GPCRs belonging to the same sequence classes can form ...

SUMMARY Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages including TLR4, TNF and type I IFN receptors are concurrently activated but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase CYLD in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While LPS-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells associated with chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). ATLL patients with aggressive subtypes have dismal outcomes. We demonstrate that ATLL cells co-opt an early checkpoint within the tumor necrosis factor receptor 1 (TNFR1) pathway, resulting in survival advantage. This early checkpoint revolves around an interaction between the deubiquitinase CYLD and its target RIPK1. The status of RIPK1 K63-ubiquitination determines cell fate by creating either a prosurvival signal (ubiquitinated RIPK1) or a death signal (deubiquitinated RIPK1). In primary ATLL samples and in cell line models, an increased baseline level of CYLD phosphorylation was observed. We therefore tested the hypothesis that this modification of CYLD, which has been reported to inhibit its deubiquitinating function, leads to increased RIPK1 ubiquitination and thus provides a prosurvival signal to ATLL cells. CYLD phosphorylation can be pharmacologically reversed by IKK inhibitors, specifically by TBK1/IKKε and IKKβ inhibitors (MRT67307 and TPCA). Both of the IKK subfamilies can phosphorylate CYLD, and the combination of MRT67307 and TPCA have a marked effect in reducing CYLD phosphorylation and triggering cell death. ATLL cells overexpressing a kinase-inactive TBK1 (TBK1-K38A) demonstrate lower CYLD phosphorylation and subsequently reduced proliferation. IKK blockade reactivates CYLD, as evidenced by the reduction in RIPK1 ubiquitination, which leads to the association of RIPK1 with the death-inducing signaling complex (DISC) to trigger cell death. In the absence of CYLD, RIPK1 ubiquitination remains elevated following IKK blockade and it does not associate with the DISC. SMAC mimetics can similarly disrupt CYLD phosphorylation and lead to ATLL cell death through reduction of RIPK1 ubiquitination, which is CYLD dependent. These results identify CYLD as a crucial regulator of ATLL survival and point to its role as a potential novel target for pharmacologic modification in this disease.