No abstract

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express μ-opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid-induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co-localized with μ-opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno-associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D-expressing mice, clozapine-N-oxide increased morphine-induced, but not vehicle-induced, locomotion. In M3D-expressing mice, clozapine-N-oxide blocked morphine-induced, but not vehicle-induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine-dependent and dopamine-independent opioid-induced rewards and locomotion.

Edited by Jeffrey E. PessinConnections between deficient autophagy and insulin resistance have emerged, however, the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO mouse embryonic fibroblasts (MEFs)), an essential autophagy gene, and observed deficient insulin and insulinlike growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of insulin receptor substrate-1 (IRS1), pivotal to insulin signaling, whereas IRS1myc overexpression recovered downstream insulin signaling. Endogenous IRS1 protein content and insulin signaling were restored in ATG16L1 KO mouse embryonic fibroblasts (MEF) upon proteasome inhibition. Through proximity-dependent biotin identification (BioID) and co-immunoprecipitation, we found that Kelch-like proteins KLHL9 and KLHL13, which together form an E3 ubiquitin (Ub) ligase complex with cullin 3 (CUL3), are novel IRS1 interactors. Expression of Klhl9 and Klhl13 was elevated in ATG16L1 KO MEFs and siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression. Moreover, Klhl13 and Cul3 knockdown increased insulin signaling. Notably, adipose tissue of high-fat fed mice displayed lower Atg16l1 mRNA expression and IRS1 protein content, and adipose tissue KLHL13 and CUL3 expression positively correlated to body mass index in humans.WeproposethatATG16L1deficiencyevokesinsulinresistance through induction of Klhl9 and Klhl13, which, in complex with Cul3, promote proteasomal IRS1 degradation.Obesity-associated type 2 diabetes (T2D), 3 one of the most prevalent diseases in Western society, is defined by whole-body insulin resistance and insufficient compensatory insulin secretion (1). In mice and humans, obesity, insulin resistance, and T2D are often associated with defects in insulin receptor substrate 1 (IRS1) (2-7). IRS1 is the direct substrate of the insulinsensing insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R). Normally, insulin-stimulated IRS1 tyrosine phosphorylation promotes phosphoinositide 3-kinase (PI3K) binding and PI3K-induced conversion of PI(4,5)P 2 to PI(3,4,5)P 3 at the cell membrane. In turn, AKT binds PI(3,4,5)P 3 via its pleckstrin homology (PH) domain, promoting AKT phosphorylation by mTOR complex 2 (mTORC2) at Ser-473 and by phosphoinosit-

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1 ) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell-to-cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin-based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3−/− mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.