Intrinsic control of cerebral blood flow in response to intravascular pressure is traditionally attributed to smooth muscle cells in arterioles. However, in this issue of , Ferris demonstrate that capillary constriction...Intrinsic control of cerebral blood flow in response to intravascular pressure is traditionally attributed to smooth muscle cells in arterioles. However, in this issue of , Ferris demonstrate that capillary constriction is caused by pressure-induced depolarization of pericytes, mural cells that encircle capillaries, and is mediated by TRPC3 cation channels, identifying the channel as critical for fine-tuning brain perfusion.
Cerebral autoregulation ensures constant blood flow, an essential condition of brain health. A fundamental parameter of the brain circulation is the dynamic regulation of microvessel diameter to allow for adjustments in...Cerebral autoregulation ensures constant blood flow, an essential condition of brain health. A fundamental parameter of the brain circulation is the dynamic regulation of microvessel diameter to allow for adjustments in resistance to blood pressure changes. Pericytes are a family of mural cells that wrap around the capillary endothelium and contribute to the dynamic control of capillary diameter. We sought to determine whether and how brain pericytes constrict in response to blood pressure elevation with in vivo two-photon microscopy, electrophysiology, and ex vivo arteriolar-capillary myography of mice with conditional mural cell knockout or with expression of a genetically encoded Ca indicator. In first- to fourth-order capillaries, pericytes displayed a rapid and measurable response to pressure by decreasing luminal diameter, depolarizing membrane potentials, and increasing cytoplasmic Ca signaling. Pharmacological and imaging approaches revealed that transient receptor potential channel 3 (TRPC3) and voltage-gated Ca channels were sequentially activated to promote fast constriction. Genetic ablation of resulted in decreased currents, loss of membrane depolarization, and near-complete ablation of the generation of tone over a standard pressure curve in transitional pericytes but not in upstream arterioles. Together, our findings identify TRPC3 channel activation as critical for proximal pericyte depolarization and contraction in response to pressure, highlighting the signaling differences between arteriolar and capillary blood flow regulation.
Morita K, Hatano A, Kokaji T
… +17 more, Sugimoto H, Tsuchiya T, Ozaki H, Egami R, Li D, Terakawa A, Ohno S, Inoue H, Inaba Y, Suzuki Y, Matsumoto M, Takahashi M, Izumi Y, Bamba T, Hirayama A, Soga T, Kuroda S
Adaptation to starvation is a multimolecular and temporally ordered process. We sought to elucidate how the healthy liver regulates various molecules in a temporally ordered manner during starvation and how obesity disru...Adaptation to starvation is a multimolecular and temporally ordered process. We sought to elucidate how the healthy liver regulates various molecules in a temporally ordered manner during starvation and how obesity disrupts this process. We used multiomic data collected from the plasma and livers of wild-type and leptin-deficient obese (/) mice at multiple time points during starvation to construct a starvation-responsive metabolic network that included responsive molecules and their regulatory relationships. Analysis of the network structure showed that in wild-type mice, the key molecules for energy homeostasis, ATP and AMP, acted as hub molecules to regulate various metabolic reactions in the network. Although neither ATP nor AMP was responsive to starvation in / mice, the structural properties of the network were maintained. In wild-type mice, the molecules in the network were temporally ordered through metabolic processes coordinated by hub molecules, including ATP and AMP, and were positively or negatively coregulated. By contrast, both temporal order and coregulation were disrupted in / mice. These results suggest that the metabolic network that responds to starvation was structurally robust but temporally disrupted by the obesity-associated loss of responsiveness of the hub molecules. In addition, we propose how obesity alters the response to intermittent fasting.
Drago-Garcia D, Giri S, Chatterjee R
… +39 more, Simoni-Nieves A, Abedrabbo M, Genna A, Rios MLU, Lindzen M, Sekar A, Gupta N, Aharoni N, Bhandari T, Mayalagu A, Schwarzmüller L, Tarade N, Zhu R, Mohan-Raju HR, Karatekin F, Roncato F, Eyal-Lubling Y, Keidar T, Nof Y, Belugali Nataraj N, Bernshtein KS, Wagner B, Nair NU, Sanghvi N, Alon R, Seger R, Pikarsky E, Donzelli S, Blandino G, Wiemann S, Lev S, Prywes R, Barkan D, Rueda OM, Caldas C, Ruppin E, Shiloh Y, Dahlhoff M, Yarden Y
Cellular plasticity mediates tissue development as well as cancer growth and progression. In breast cancer, a shift to a more epithelial phenotype (epithelialization) underlies a state of reversible cell growth arrest ca...Cellular plasticity mediates tissue development as well as cancer growth and progression. In breast cancer, a shift to a more epithelial phenotype (epithelialization) underlies a state of reversible cell growth arrest called tumor dormancy, which enables drug resistance, tumor recurrence, and metastasis. Here, we explored the mechanisms driving epithelialization and dormancy in aggressive mesenchymal-like breast cancer cells in three-dimensional cultures. Overexpressing either of the epithelial lineage-associated transcription factors OVOL1 or OVOL2 suppressed cell proliferation and migration and promoted transition to an epithelial morphology. The expression of (and of to a lesser extent) was regulated by steroid hormones and growth factors and was more abundant in tumors than in normal mammary cells. An uncharacterized and indirect target of OVOL1/2, , exhibited genetic and epigenetic aberrations in breast tumors, and its expression correlated with poor prognosis in patients. We further found that C1ORF116 was an autophagy receptor that directed the degradation of antioxidant proteins, including thioredoxin. Through C1ORF116 and unidentified mediators, OVOL1 expression dysregulated both redox homeostasis (in association with increased ROS, decreased glutathione, and redistribution of the transcription factor NRF2) and DNA damage and repair (in association with increased DNA oxidation and double-strand breaks and an altered interplay among the kinases p38-MAPK, ATM, and others). Because these effects, as they accumulate in cells, can promote metastasis and dormancy escape, the findings suggest that OVOLs not only promote dormancy entry and maintenance in breast cancer but also may ultimately drive dormancy exit and tumor recurrence.
A blended proteomics platform provides new depth to the proteome of breast cancer.A blended proteomics platform provides new depth to the proteome of breast cancer.
Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, presents clinical challenges. Unlike adrenergic (ADRN) neuroblastoma cells, neuroblastoma cells with a mes...Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, presents clinical challenges. Unlike adrenergic (ADRN) neuroblastoma cells, neuroblastoma cells with a mesenchymal (MES) identity are resistant to chemotherapy and retinoid therapy, which contributes to relapses and treatment failures. We explored whether up-regulation of the neurogenic, tumor suppressor microRNA miR-124 could promote the differentiation of retinoic acid-resistant MES neuroblastoma cells. Leveraging our screen for miRNA-modulatory small molecules, we identified and validated the tyrosine and phosphoinositide kinase inhibitor PP121 as a robust inducer of miR-124. Combining PP121 and BDNF-activating bufalin synergistically arrested proliferation and promoted the sustained differentiation of MES/heterogeneous SK-N-AS cells over several weeks. This protocol also resulted in the differentiation of multiple MES neuroblastoma and glioblastoma cell lines. RNA-seq analysis of differentiated MES/heterogeneous SK-N-AS cells revealed the replacement of the ADRN core regulatory circuitry with circuitries associated with chromaffin cells and Schwann cell precursors. Furthermore, differentiation was associated with inhibition of the CDK4/CDK6 pathway and activation of a transcriptional program that correlated with improved outcomes for patients with neuroblastoma. Our findings suggest an approach with translational potential to induce the differentiation of therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells could be used to study mechanisms underlying cancer biology and therapies.
Covalent protein modification with endogenously produced cyanide supports mammalian cell proliferation.Covalent protein modification with endogenously produced cyanide supports mammalian cell proliferation.
The developmental sculpting of neuromuscular circuitry in early postnatal life occurs through the process of synapse elimination: Supernumerary axon inputs are gradually eliminated from the neuromuscular junction (NMJ),...The developmental sculpting of neuromuscular circuitry in early postnatal life occurs through the process of synapse elimination: Supernumerary axon inputs are gradually eliminated from the neuromuscular junction (NMJ), resulting in each muscle fiber being innervated by a single axon terminal. Here, we investigated the molecular pathways underlying this process using a ChAT-RiboTag mouse model in which we isolated ribosome-bound mRNAs in motor neurons during synapse elimination in vivo. Analysis of these mRNAs using translating ribosome affinity purification followed by RNA sequencing (TRAP-seq) revealed dynamic changes in the motor neuron translatome over the first 2 weeks of life, which were largely independent of parallel transcriptional changes and correlated with the progressive elimination of supernumerary inputs. Bioinformatic analysis identified distinct clusters of transcripts that were translated at specific time points during synapse elimination. Treating mice with two small molecules that were predicted to independently target the proteins or pathways encoded by the transcript cluster associated with neural metabolism increased the rate of synapse elimination in vivo. Together, these data provide a cell type-specific overview of temporal modifications occurring in the motor neuron translatome during synapse elimination, revealing rapid and dynamic responses to postnatal developmental cues.
Sleep loss dysregulates cellular metabolism and energy homeostasis. Highly metabolically active cells, such as neurons, enter a catabolic state during periods of sleep loss, which consequently disrupts physiological func...Sleep loss dysregulates cellular metabolism and energy homeostasis. Highly metabolically active cells, such as neurons, enter a catabolic state during periods of sleep loss, which consequently disrupts physiological functioning. Specific to the central nervous system, sleep loss results in impaired synaptogenesis and long-term memory, effects that are also characteristic of neurodegenerative diseases. In this review, we describe how sleep deprivation increases resting energy expenditure, leading to the development of a negative energy balance-a state with insufficient metabolic resources to support energy expenditure-in highly active cells like neurons. This disruption of energetic homeostasis alters the balance of metabolites, including adenosine, lactate, and lipid peroxides, such that energetically costly processes, such as synapse formation, are attenuated. During sleep loss, metabolically active cells shunt energetic resources away from those processes that are not acutely essential, like memory formation, to support cell survival. Ultimately, these findings characterize sleep loss as a metabolic disorder.
Stimulating efferocytosis, the phagocytic removal of apoptotic cells by macrophages, has been proposed as a method to eliminate dying or dead cells that accumulate and contribute to diseases such as cancer, atheroscleros...Stimulating efferocytosis, the phagocytic removal of apoptotic cells by macrophages, has been proposed as a method to eliminate dying or dead cells that accumulate and contribute to diseases such as cancer, atherosclerosis, and infection. Toxicity related to the off-target clearance of healthy tissue has led to the premature termination of multiple clinical programs for proefferocytic therapies. To identify potential proefferocytic therapies with established risk profiles, we screened ~3000 US Food and Drug Administration (FDA)-approved drugs and other well-characterized compounds for their capacity to stimulate efferocytosis. We found that the antipsychotic drug thiothixene stimulated efferocytosis of apoptotic and lipid-laden cells by mouse and human macrophages and enhanced the continual efferocytosis of apoptotic cells. Consistent with thiothixene's suppressive effects on dopaminergic signaling, dopamine potently inhibited efferocytosis in a manner that was only partially reversed by thiothixene. The prophagocytic effects of thiothixene in mouse macrophages depended on increased expression of the gene encoding the retinol-binding protein receptor Stra6L, which, in turn, promoted the production of the continual efferocytosis stimulator arginase 1. Our findings demonstrate that dopamine inhibits efferocytosis in macrophages and identify thiothixene, a generic, FDA-approved antipsychotic drug that has been in use for more than 50 years, as a promising candidate for promoting continual efferocytosis and the removal of diseased tissue.
Neurons are postmitotic, nonregenerative cells that have evolved fine-tuned immunological responses to maintain life-long cellular integrity, including resistance to common programmed cell death pathways such as necropto...Neurons are postmitotic, nonregenerative cells that have evolved fine-tuned immunological responses to maintain life-long cellular integrity, including resistance to common programmed cell death pathways such as necroptosis. We previously demonstrated a necroptosis-independent role for the key necroptotic kinase RIPK3 in host defense against neurotropic flavivirus infection. Here, we show that RIPK3 activation had distinct outcomes in primary cortical neurons when compared with mouse embryonic fibroblasts (MEFs) during Zika virus (ZIKV) infection or after sterile activation. We found that RIPK3 activation did not induce neuronal death but instead drove antiviral gene transcription after ZIKV infection. Although RIPK3 activation in MEFs induced cell death, ablation of downstream cell death effectors unveiled a RIPK3-dependent transcriptional program that largely overlapped with that observed in ZIKV-infected neurons. In death-resistant MEFs, RIPK3-dependent transcription relied on interactions with the RHIM domain-containing proteins RIPK1 and TRIF, similar to the requirements for the RIPK3-dependent antiviral transcriptional signature in ZIKV-infected neurons. These findings suggest that the pleotropic functions of RIPK3 are largely context dependent and that in cells that are resistant to cell death, RIPK3 acts as a mediator of inflammatory transcription.
A bacterium in the maternal gut promotes stem cell activity and long-term gut and brain health in offspring.A bacterium in the maternal gut promotes stem cell activity and long-term gut and brain health in offspring.
Human embryonic carcinoma (hEC) cells are derived from teratocarcinomas, exhibit robust proliferation, have a high differentiation potential, are the malignant counterparts of human embryonic stem cells (hESCs), and are...Human embryonic carcinoma (hEC) cells are derived from teratocarcinomas, exhibit robust proliferation, have a high differentiation potential, are the malignant counterparts of human embryonic stem cells (hESCs), and are considered hESC-like. The chromosomal passenger complex (CPC), made up of the microtuble binding protein Borealin, the kinase Aurora-B, the CPC-stabilizing inner centromere protein (INCENP), and the inhibitor of apoptosis family member Survivin, regulates cell division and is active exclusively during mitosis in somatic cells. The anaphase-promoting complex/cyclosome and its cofactor Cdh1 (APC/C) is a ubiquitylating complex that catalyzes the degradation of Aurora-B and Borealin in somatic cells but has low activity during interphase in hESCs. Here, we found that Borealin and Aurora-B exhibited sustained stability throughout the cell cycle of hEC cells due to low APC/C activity. In contrast with somatic cells, CPC activity persisted across the cell cycle of hEC cells because of diminished APC/C activity. Disrupting the CPC complex by depleting its constituents triggered spontaneous differentiation in hEC cells. As hEC cells differentiated, APC/C activation curtailed CPC activity. Inactivating the CPC by pharmacologically inhibiting Aurora-B induced hEC cell differentiation by activating the epithelial-to-mesenchymal transition (EMT) program. Hence, APC/C-mediated termination of CPC activity triggered hEC cell differentiation. Collectively, these findings demonstrate a role for the CPC in governing hESC cell fate.
The canonical model of G protein-coupled receptor (GPCR) signaling comprises G protein activation at the plasma membrane, followed by receptor phosphorylation and β-arrestin recruitment, which leads to receptor desensiti...The canonical model of G protein-coupled receptor (GPCR) signaling comprises G protein activation at the plasma membrane, followed by receptor phosphorylation and β-arrestin recruitment, which leads to receptor desensitization and endocytosis. However, the activation of some GPCRs results in sustained G protein signaling from intracellular compartments in a manner reportedly dependent on β-arrestin and receptor endocytosis. The vasopressin type 2 receptor (VR) can be activated by two structurally similar hormones, arginine vasopressin and oxytocin, both of which stimulate the production of the second messenger cyclic adenosine monophosphate (cAMP). In this study, we showed that sustained VR signaling and endosomal Gα (stimulatory G protein alpha subunit) translocation could occur without β-arrestin-mediated receptor endocytosis and was primarily controlled by the residence time of the ligand on the receptor. β-Arrestin had opposing effects on sustained signaling: It facilitated receptor internalization into endosomes, where it activated Gα, and promoted cAMP production from this compartment. However, β-arrestin-mediated receptor endocytosis also induced ligand dissociation due to the acidic endosomal environment, thereby limiting the signal. Overall, our data suggest that signals originating at the plasma membrane play a dominant role in sustained VR signaling stimulated by arginine vasopressin.
The activation of heterotrimeric G proteins (Gαβγ) by G protein-coupled receptors (GPCRs) is a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane and a target for many clinical drugs. Ma...The activation of heterotrimeric G proteins (Gαβγ) by G protein-coupled receptors (GPCRs) is a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane and a target for many clinical drugs. Many optical biosensors commonly used for measuring GPCR-stimulated G protein activity rely on exogenously expressed GPCRs and/or G proteins, which compromise readout fidelity. Biosensors that measure endogenous signaling may interfere with the signaling process under investigation or have a limited dynamic range of detection, hindering applicability. Here, we developed an optical BRET-based biosensor, Gα bONE-GO, that detects endogenous GTP-bound (active) Gα upon stimulation of endogenous GPCRs more robustly than existing sensors of endogenous activity. Its design leverages the Gα-binding protein GINIP as a high-affinity and specific detector of Gα-GTP. We optimized this design to prevent interference with downstream G-dependent signaling and to enable implementation in different experimental systems having endogenous GPCRs, including adenosine receptors in primary astroglial cells and opioid receptors in cell lines. In a neuronal cell line, Gα bONE-GO revealed activation profiles indicating that several natural opioid neuropeptides acted as partial agonists, in contrast with their characterization as full agonists using biosensors that depend on exogenously expressed receptors and G proteins. The Gα bONE-GO biosensor is a direct and sensitive detector of endogenous activation of Gα proteins by GPCRs in different experimental settings but does not interfere with the subsequent propagation of signaling.
Chen X, Tang X, Xie Y
… +13 more, Cuffari BJ, Tang C, Cao F, Gao X, Meng Z, Noble PW, Young MR, Turk OM, Shirali A, Gera J, Nishimura RN, Zhou J, Hansen JE
Nucleic acid-mediated signaling triggers an immune response that is believed to be central to the pathophysiology of autoimmunity in systemic lupus erythematosus (SLE). Here, we found that a cell-penetrating, SLE-associa...Nucleic acid-mediated signaling triggers an immune response that is believed to be central to the pathophysiology of autoimmunity in systemic lupus erythematosus (SLE). Here, we found that a cell-penetrating, SLE-associated antiguanosine autoantibody may present therapeutic opportunities for cancer treatment. The autoantibody entered cells through a nucleoside salvage-linked pathway of membrane transit that avoids endosomes and lysosomes and bound to endogenous RNA in live cells. In orthotopic models of glioblastoma, the antibody localized to areas adjacent to necrotic tumor cells and promoted animal survival in a manner that depended on T cells. Mechanistic studies revealed that antibody binding to nucleic acids activated the cytoplasmic pattern recognition receptor cyclic GMP-AMP synthase (cGAS), thereby stimulating immune signaling and cGAS-dependent cytotoxicity. Moreover, the autoantibody could carry and deliver functional RNA into tumor, brain, and muscle tissues in live mice when administered locally. The findings establish a collaborative autoantibody-nucleic acid interaction that is translatable to strategies for nonviral gene delivery and immunotherapy.
An antibody delivered nasally after brain injury induces a neuroprotective, anti-inflammatory response.An antibody delivered nasally after brain injury induces a neuroprotective, anti-inflammatory response.
A synthetic cannabinoid biases CB1 signaling toward G proteins to elicit effective pain relief without tolerance in mice.A synthetic cannabinoid biases CB1 signaling toward G proteins to elicit effective pain relief without tolerance in mice.