The highly addictive psychostimulant methamphetamine increases the release of dopamine in the brain's reward circuitry, where it also promotes the release of cytokines, including TNF-α, that contribute to neuroinflammation associated with methamphetamine abuse. Here, we found a dynamic interplay between methamphetamine and TNF-α in facilitating dopamine transmission within the ventral tegmental area (VTA) in mice. In ex vivo mouse brain slices and dopaminergic neurons, methamphetamine or TNF-α treatment increased dopamine release, intracellular Ca2+ concentrations, and the firing activity of VTA dopaminergic neurons. These effects depended on the activity of dopamine transporter (DAT) and L-type voltage-gated Ca2+ channels. Pharmacological inhibition of either DAT or TNF-α signaling mitigated these effects, suggesting that methamphetamine-induced alterations in VTA dopaminergic neurons are partially TNF-α dependent. These results underscore the role of neuroimmune signaling in modulating the dopaminergic circuitry and may inform therapeutic strategies for addressing methamphetamine addiction and its associated neuroinflammatory disorders.

Dopamine drives the neuronal activity and synaptic plasticity required for various forms of learning. It supports short-term working memory through activation of the D1-like dopamine receptors D1 and D5. Here, we found that the L-type Ca2+ channel CaV1.2 was a critical mediator of D1/5 signaling in hippocampal pyramidal neurons. In cultured mouse hippocampal neurons, a D1/5 agonist augmented currents through CaV1.2 in the soma and Ca2+ influx in the dendrites. This effect was mediated through the second messenger cAMP and cAMP-dependent protein kinase (PKA), which phosphorylated the CaV1.2 α1 subunit at Ser1928. CaV1.2 and D5 colocalized, suggesting that this signaling was spatially restricted. In mice, D1/5 agonism facilitated spatial working memory in wild-type but not litter-matched CaV1.2 α1 S1928A knockin animals. These findings identify CaV1.2 as a key D1/5 signaling effector that supports dopamine-driven executive functions in cognition.

Blocking the cell cycle is a promising avenue for cancer therapy, with cyclin-dependent kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, the activities of CDK4 and CDK6 (CDK4/6) compensate for CDK2 inhibition and sustain tumor cell proliferation, enabling CDK2 reactivation. Thus, we hypothesized that sensitivity to CDK2 inhibition is linked to the absence of this CDK4/6-mediated compensatory mechanism. We found that cyclin E1–driven ovarian cancers often coexpressed the tumor suppressor p16, which inhibited CDK4/6 signaling. Single-cell time-lapse imaging showed that high abundance of p16 conferred increased sensitivity to CDK2 inhibitors, whereas depletion of p16 rendered cells more resistant to CDK2 inhibition through CDK4/6-dependent compensation. Concordantly, acquired resistance to CDK2 inhibitors correlated with reduced p16 and increased cyclin D1 protein abundance. Multiplexed immunofluorescence of 225 ovarian tumors from patients revealed that 18% of the tumors had high cyclin E1 and p16 expression. Thus, p16 may be a useful biomarker for identifying patients most likely to benefit from CDK2 inhibitors.

The α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) in G protein-coupled receptor (GPCR) pathways operate as gate switches by cycling through inactive GDP-bound and active GTP-bound states. Three flexible regions (switches I to III) of Gα subunits undergo the most substantial conformational rearrangements and determine interactions with receptors, signaling regulators, and effectors. Here, we describe three patients with severe pediatric encephalopathy harboring c.723+1G>A or c.723+2T>A variants in the splicing donor site of intron 6 of GNAO1, the gene encoding the major neuronal G protein α subunit Gαo. These substitutions destroyed the conserved GU sequence of the pre-mRNA and rendered the donor site unrecognizable, prompting cryptic splice site engagement and production of the dominant pathogenic Gαo[V234_T241del] variant, which lacked switch III (amino acid residues 232 to 243 in Gαo). Structural, biochemical, and cellular analyses characterized V234_T241del as a strong neomorphic variant that was severely deficient in guanine nucleotide handling and cellular interactions and sensitive to zinc salts, an emerging targeted treatment for a GNAO1 encephalopathy subgroup. Our studies provide pathological and molecular insights into the function and effect of removal of an entire switch region in 1 of the 16 human G protein α subunits that underlie multiple physiological processes and diverse pathologies.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is linked to a shift in the composition of the gut microbiome. Here, we found that depletion of the gut microbiome in adult zebrafish led to the development of steatotic liver disease in animals on standard diets. Using metabolomics and isotope tracing, we found that dietary glucose was converted to sorbitol by host intestinal cells. Although bacteria degraded sorbitol in control animals, sorbitol was transferred to the livers of fish when the gut microbiome had been depleted. Within the liver, sorbitol was converted into fructose 1-phosphate, which subsequently activated glucokinase and increased glycolytic flux, leading to increased hepatic glycogen and fat content. Inhibition of sorbitol production in microbiome-depleted animals was sufficient to prevent the development of steatotic liver, and colonizing the intestines of microbiome-depleted fish with sorbitol-degrading Aeromonas bacterial strains rescued the steatotic liver phenotype. Conversely, exogenous administration of high concentrations of sorbitol phenocopied gut microbiota depletion and induced hepatic steatosis. Together, these findings show that sorbitol-degrading bacteria in the gut protect against steatotic liver disease and suggest that excessive intake of dietary sorbitol may pose a risk for the development of MASLD.

Coordinated posttranslational modifications of cysteine residues regulate STING oligomerization and function.

Ryanodine receptor 1 (RYR1) is the sarcoplasmic reticulum (SR) Ca2+ release channel required for both skeletal muscle contraction and Ca2+ leak. Mutations in RYR1 cause malignant hyperthermia susceptibility (MHS) and enhanced sensitivity to heat stroke (ESHS), which can result in death due to excessive skeletal muscle thermogenesis upon exposure to volatile anesthetics or heat. Here, we investigated the molecular and physiological functions of phosphorylation of RYR1 at Ser2902 by the kinase striated muscle preferentially expressed protein (SPEG). Muscle from SPEG-deficient mice expressing RYR1 with a Ser2902→Asp2902 (S2902D) point mutation to mimic phosphorylation by SPEG showed decreased SR Ca2+ sparks. Muscle from mice homozygous for the S2902D point mutation had reduced SR Ca2+ transients and small changes in force generation but overall mild phenotypic changes. YS mice, which are heterozygous for a Tyr524→Ser524 point mutation in RYR1, show increased Ca2+ leak and are a model of MHS and ESHS. Crossing YS mice with S2902D mice led to decreased SR Ca2+ leak and desensitized the mice to both volatile anesthetics and heat. Thus, SPEG inhibits SR Ca2+ leak in skeletal muscle by phosphorylating Ser2902 on RYR1, and mutation of Ser2902 to Asp2902 to mimic this phosphorylation event rescues YS mice from heat-induced death.

In BRAF-mutant melanoma cells treated with inhibitors of the kinases BRAF and MEK, a subset of cells rapidly and nongenetically adapts to escape drug-induced quiescence and reenters the cell cycle. Here, we investigated the mechanisms enabling this drug escape by computationally reconstructing single-cell lineages from time-lapse imaging data, linking dynamic signaling pathways to distinct cell-cycle fate outcomes. We found that reactivation of the MEK substrate ERK was necessary but not sufficient to drive escape; rather, the activity of the protein complex mTORC1 was also required to promote cell growth and protein synthesis in drug-treated cells destined for cell-cycle reentry. ERK and mTORC1 signaling converged to increase the abundance of cyclin D1 protein, a critical bottleneck for cell-cycle commitment under drug pressure. In cells in which endogenous cyclin D1 was fluorescently tagged using CRISPR, the subset that escaped drug treatment exhibited marked accumulation of cyclin D1 at least 15 hours before cell-cycle reentry, enabling early prediction of future drug escape. Cyclin D1 thus represents both an early biomarker and potential therapeutic target for suppressing drug escape in melanoma. We observed a similar mTORC1-driven mechanism underlying escape in lung cancer cells, but not colon cancer cells, highlighting partial generalizability across cancer types.

Positive allosteric modulators of free fatty acid receptor 2 induce distinct conformations to bias G protein signaling.

Small extracellular vesicles (sEVs) released from tumors recruit nociceptor neurons to the tumor bed. Here, we found that ablating these neurons in mouse models of head and neck carcinoma and melanoma reduced the infiltration of myeloid-derived suppressor cells (MDSCs). Moreover, sEV-deficient tumors failed to develop in mice lacking nociceptor neurons. We investigated the interplay between tumor-infiltrating nociceptors and immune cells in head and neck squamous cell carcinoma (HNSCC) and melanoma. Upon exposure to cancer-derived sEVs, mouse dorsal root ganglion (DRG) neurons secreted increased amounts of substance P, IL-6, and injury-associated neuronal markers. Patient-derived sEVs sensitized DRG neurons to capsaicin, implying enhanced nociceptor responsiveness. Furthermore, nociceptors cultured with sEVs induced an immunosuppressed state in CD8+ T cells. Incubation with conditioned medium from cocultures of neurons and cancer cells resulted in increased expression of markers of MDSCs and suppressive function in primary bone marrow cells, and the combination of neuron-conditioned medium and cancer sEVs promoted checkpoint receptor expression on T cells. Together, these findings reveal that nociceptor neurons facilitate CD8+ T cell exhaustion and bolster MDSC infiltration into HNSCC and melanoma. Consequently, targeting nociceptors may provide a strategy to disrupt detrimental neuroimmune cross-talk in cancer and potentiate antitumor immunity.