Receptor-independent Mechanism Research Articles

Protection of HT22 neuronal cells against chemically-induced ferroptosis by catechol estrogens: protein disulfide isomerase as a mechanistic target

Ferroptosis is a form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Biochemically, ferroptosis can be selectively induced by erastin through glutathione depletion or through inhibition of glutathione peroxidase 4 by RSL3, which leads to accumulation of cytotoxic lipid reactive oxygen species (ROS). Protein disulfide isomerase (PDI) was recently shown to mediate erastin/RSL3-induced ferroptosis and thus also become a new target for protection against chemically-induced ferroptosis. The present study aims to identify endogenous compounds that can protect against erastin/RSL3-induced ferroptotic cell death. We find that 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone and 4-hydroxyestradiol, four major endogenous catechol estrogens, are effective inhibitors of PDI, and can strongly protect against chemically-induced ferroptotic cell death in cultured HT22 mouse hippocampal neuronal cells. The CETSA assay showed that these catechol estrogens can bind to PDI in live cells. PDI knockdown attenuates the protective effect of these catechol estrogens against chemically-induced ferroptosis. Mechanistically, inhibition of PDI’s catalytic activity by catechol estrogens abrogates erastin/RSL3-induced dimerization of nitric oxide synthase, thereby preventing the subsequent accumulation of cellular nitric oxide, ROS and lipid-ROS, and ultimately ferroptotic cell death. In addition, joint treatment of cells with catechol estrogens also abrogates erastin/RSL3-induced upregulation of nitric oxide synthase protein levels, which also contributes to the cytoprotective effect of the catechol estrogens. In conclusion, the present study demonstrates that the catechol estrogens are protectors of HT22 neuronal cells against chemically-induced ferroptosis, and inhibition of PDI’s catalytic activity by these estrogens contributes to a novel, estrogen receptor-independent mechanism of cytoprotection.

Raloxifene Prevents Chemically-Induced Ferroptotic Neuronal Death In Vitro and In Vivo.

Ferroptosis, a regulated form of cell death characterized by excessive iron-dependent lipid peroxidation, can be readily induced in cultured cells by chemicals such as erastin and RSL3. Protein disulfide isomerase (PDI) has been identified as an upstream mediator of chemically induced ferroptosis and also a target for ferroptosis protection. In this study, we discovered that raloxifene (RAL), a selective estrogen receptor modulator known for its neuroprotective actions in humans, can effectively inhibit PDI function and provide robust protection against chemically induced ferroptosis in cultured HT22 neuronal cells. Specifically, RAL can bind directly to PDI both in vitro and in intact neuronal cells and inhibit its catalytic activity. Computational modeling analysis reveals that RAL can tightly bind to PDI through forming a hydrogen bond with its His256 residue, and biochemical analysis further shows that when PDI's His256 is mutated to Ala256, RAL loses its inhibition of PDI's catalytic activity. This inhibition of PDI by RAL significantly reduces the dimerization of both the inducible and neuronal nitric oxide synthases and the accumulation of nitric oxide, both of which have recently been shown to play a crucial role in mediating chemically induced ferroptosis through subsequent induction of ROS and lipid-ROS accumulation. In vivo behavioral analysis shows that mice treated with RAL are strongly protected against kainic acid-induced memory deficits and hippocampal neuronal damage. In conclusion, this study demonstrates that RAL is a potent inhibitor of PDI and can effectively prevent chemically induced ferroptosis in hippocampal neurons both in vitro and in vivo. These findings offer a novel estrogen receptor-independent mechanism for RAL's neuroprotective actions in animal models and humans.

Serotonylation in tumor-associated fibroblasts contributes to the tumor-promoting roles of serotonin in colorectal cancer

Accumulated studies have highlighted the diverse roles of 5-hydroxytryptamine (5-HT), or serotonin, in cancer biology, particularly in colorectal cancer (CRC). While 5-HT primarily exerts its effects through binding to various 5-HT receptors, receptor-independent mechanisms such as serotonylation remain unclear. This study revealed that depleting 5-HT, either through genetic silencing of Tph1 or using a selective TPH1 inhibitor, effectively reduced the growth of CRC tumors. Interestingly, although intrinsic 5-HT synthesis exists in CRC, it is circulating 5-HT that mediates the cancer-promoting function of 5-HT. Blocking the function of 5-HT receptors showed that the oncogenic roles of 5-HT in CRC operate through a mechanism that is separate from its receptor. Instead, serotonylation of histone H3Q5 (H3Q5ser) was found in CRC cells and cancer-associated fibroblasts (CAFs). H3Q5ser triggers a phenotypic switch of CAFs towards an inflammatory-like CAF (iCAF) subtype, which further enhances CRC cell proliferation, invasive characteristics, and macrophage polarization. Knockdown of the 5-HT transporter SLC22A3 or inhibition of TGM2 reduces H3Q5ser levels and reverses the tumor-promoting phenotypes of CAFs in CRC. Collectively, this study sheds light on the serotonylation-dependent mechanisms of 5-HT in CRC progression, offering insights into potential therapeutic strategies targeting the serotonin pathway for CRC treatment.

MULTIDRUG RESISTANT DYSLIPIDEMIA: A CASE REPORT

Abstract Introduction Recently introduced lipid–lowering agents (PCSK9–i, bempedoic acid) can efficiently treat statin–intolerant patients, who couldn‘t reach LDL target before. Nonetheless, there are still cases where the pharmacological management may have strong limitations. Case Report A 71 year–old woman, with multiple cardiovascular risk factors (diabetes, hypertension and familial dylipidemia, LDL–C 276) underwent heart transplantation for a hypertrophic cardiomyopathy with an evolution towards a dilated phenotype in 2005. Before the transplant, she was on low–dose statin treatment (pravastatin 10 mg/die) that was suspended years later owing to hyper–CKemia (600 U/L), despite her being totally asymptomatic. A second attempt with simvastatin/ezetimibe 40/10 mg resulted again in elevation of CK levels, associated with significant muscular pain. Thus, just ezetimibe was continued. In 2020, 15 years after the transplant, the patient underwent a coronary angiography showing a moderate coronary allograft vasculopathy (ISHLT CAV2). Specifically, a sub occlusive ostial left main coronary artery lesion was treated by percutaneous angioplasty and stent implantation. Since a lower LDL target was needed (LDL–C 55 mg/dL), Alirocumab was introduced. However, 6 months later, no significant LDL levels reduction had been observed. The shift to evolocumab led to the same result, although the patient reported full therapeutic compliance. In the suspicion of anti–drug antibodies development, we opted for Inclisiran without achieving a significant decrease in LDL–C levels. In parallel with the search for genetic mutations in the PCSK9 gene and other genes involved in cholesterol metabolism (including LDLR, APOB, and LDLRAP1 and resulting positive only for common variants in LDLR receptor), therapy with bempedoic acid was initiated, again without substantial benefit (LDL–C 118 mg/dL). Conclusion Despite cholesterol management has been simplified by the introduction of new drugs, it may still be complex in patients requiring very low LDL–C levels in some specific cases. Drugs with a LDL receptor–independent mechanism of action (e.g., evinacumab) may allow the achievement of therapeutic goals suggested by clinical guidelines.

MULTIDRUG RESISTANT DYSLIPIDEMIA: A CASE REPORT

Objective: Recently introduced lipid-lowering agents (PCSK9-i, bempedoic acid) can efficiently treat statin-intolerant patients, who couldn’t reach LDL target before. Nonetheless, there are still cases where the pharmacological management may have strong limitations. Design and method: A 71 year-old woman, with multiple cardiovascular risk factors (diabetes, hypertension and familial dylipidemia, LDL-C 276) underwent heart transplantation for a hypertrophic cardiomyopathy with an evolution towards a dilated phenotype in 2005. Before the transplant, she was on low-dose statin treatment (pravastatin 10 mg/die) that was suspended years later owing to hyper-CKemia (600 U/L), despite her being totally asymptomatic. A second attempt with simvastatin/ezetimibe 40/10 mg resulted again in elevation of CK levels, associated with significant muscular pain. Thus, just ezetimibe was continued. In 2020, 15 years after the transplant, the patient underwent a coronary angiography showing a moderate coronary allograft vasculopathy (ISHLT CAV2). Specifically, a sub occlusive ostial left main coronary artery lesion was treated by percutaneous angioplasty and stent implantation. Since a lower LDL target was needed (LDL-C 55 mg/dL), Results: Alirocumab was introduced. However, 6 months later, no significant LDL levels reduction had been observed. The shift to evolocumab led to the same result, although the patient reported full therapeutic compliance. In the suspicion of anti-drug antibodies development, we opted for Inclisiran without achieving a significant decrease in LDL-C levels. In parallel with the search for genetic mutations in the PCSK9 gene and other genes involved in cholesterol metabolism (including LDLR, APOB, and LDLRAP1 and resulting positive only for common variants in LDLR receptor), therapy with bempedoic acid was initiated, again without substantial benefit (LDL-C 118 mg/dL). Conclusions: Despite cholesterol management has been simplified by the introduction of new drugs, it may still be complex in patients requiring very low LDL-C levels in some specific cases. Drugs with a LDL receptor-independent mechanism of action (e.g., evinacumab) may allow the achievement of therapeutic goals suggested by clinical guidelines.

B-cell–directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates

AbstractChimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell–derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell–targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor–independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell–targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Abstract 496: Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis

Abstract Gold nanoparticles for therapeutic interventions have become a widely popular source due to the ease in surface chemistry available for modification. Several studies undertaken focusses on the size, shape of nanoparticles as well as the signaling mechanisms and yet, knowledge regarding the alteration in membrane dynamics such as nanomechanical properties and cytokines involved during their uptake has not been studies before. To overcome the knowledge deficit, we employed atomic force microscopy and qRT-PCR techniques to acquire nanomechanical attributes and cytokines expressions respectively, in Panc-1 and AsPC-1 cell lines treated with surface modified gold nanoparticles for 1- and 24-hours’ time point. In nanomechanics paradigm, impetus was given on both linear attributes such as membrane stiffness, deformation, and adhesion as well as non-linear attributes such as drained Poisson’s ratio, diffusion coefficient and pore size. On the other hand, qRT-PCR technique yielded alterations in relative mRNA expressions of pro-tumorigenic cytokines such as CCL2, CXCL1, CXCL2, CXCL3, IL8, IL11, IL18 and TGFβ-1. Further, using Pearson’s correlation, we deduced the dependency between nanomechanical properties and cytokines expressions. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and 1 hr vs 24hrs, to deduce correlations between cytokines and nanomechanical attributes. In Panc-1 cell line, we observed that PEGylation vs non-PEGylation of gold nanoparticles demonstrated most significant correlations, whereas in AsPC-1 cell line, receptor independent mechanism played a pivotal role in establishing significant correlations. For instance, D_C and P_D were highly correlated with CXCL1, CXCL2 and TGFβ-1 when non-PEGylated gold nanoparticles were up taken by Panc-1 cells. Similarly, D_C and P_D were highly correlated consistently with our cytokine panel except for TGFβ-1. In AsPC-1 cell line, deformation was significantly correlated with CCL2, CXCL2, CXCL3 and IL-8 whereas, D_C and P_D were significantly correlated with CCL2, CXCL1, CXCL3, IL-18 and TGFβ-1 in receptor independent scenario. This study demonstrated that during the uptake of gold nanoparticles, non-linear nanomechanical attributes are detrimental over linear attributes. Moreover, Surface modified gold nanoparticles induce varying levels of alteration in pro-tumorigenic cytokines. Taken together, this study boosts our insights into correlations describing cytokines regulating nanomechanical attributes in endocytosis paradigm. Citation Format: Tanmay Kulkarni, Enfeng Wang, Ramcharan Singh Angom, Debabrata Mukhopadhyay, Santanu Bhattacharya. Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 496.

Significance of Melatonin in the Regulation of Circadian Rhythms and Disease Management.

Melatonin, the 'hormone of darkness' is a neuronal hormone secreted by the pineal gland and other extra pineal sites. Responsible for the circadian rhythm and seasonal behaviour of vertebrates and mammals, melatonin is responsible for regulating various physiological conditions and the maintenance of sleep, body weight and the neuronal activities of the ocular sites. With its unique amphiphilic structure, melatonin can cross the cellular barriers and elucidate its activities in the subcellular components, including mitochondria. Melatonin is a potential scavenger of oxygen and nitrogen-reactive species and can directly obliterate the ROS and RNS by a receptor-independent mechanism. It can also regulate the pro- and anti-inflammatory cytokines in various pathological conditions and exhibit therapeutic activities against neurodegenerative, psychiatric disorders and cancer. Melatonin is also found to show its effects on major organs, particularly the brain, liver and heart, and also imparts a role in the modulation of the immune system. Thus, melatonin is a multifaceted candidate with immense therapeutic potential and is still considered an effective supplement on various therapies. This is primarily due to rectification of aberrant circadian rhythm by improvement of sleep quality associated with risk development of neurodegenerative, cognitive, cardiovascular and other metabolic disorders, thereby enhancing the quality of life.

The role of proprotein convertase subtilisin/kexin 9 (PCSK9) in macrophage activation: a focus on its LDL receptor-independent mechanisms.

Recent clinical trials demonstrated that proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors reduce cardiovascular events without affecting systemic inflammation in the patients with coronary artery disease, as determined by high sensitivity C-reactive protein (CRP) levels. However, its pro-inflammatory effects in cardiovascular disease in humans and experimental animals beyond the traditional cholesterol receptor-dependent lipid metabolism have also called attention of the scientific community. PCSK9 may target receptors associated with inflammation other than the low-density lipoprotein receptor (LDLR) and members of the LDLR family. Accumulating evidence suggests that PCSK9 promotes macrophage activation not only via lipid-dependent mechanisms, but also lipid-independent and LDLR-dependent or -independent mechanisms. In addition to dyslipidemia, PCSK9 may thus be a potential therapeutic target for various pro-inflammatory diseases.

Tubular albumin uptake: is there evidence for a quantitatively important, receptor-independent mechanism?

Tubular albumin uptake: is there evidence for a quantitatively important, receptor-independent mechanism?

Melanogenesis Is Directly Affected by Metabolites of Melatonin in Human Melanoma Cells.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic (N1-acetyl-N2-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients.

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Anti-hormone therapies are not efficacious for reducing the incidence of triple negative breast cancer (TNBC), which lacks both estrogen and progesterone receptors. While the etiology of this aggressive breast cancer subtype is unclear, visceral obesity is a strong risk factor for both pre- and post-menopausal cases. The mechanism by which excessive deposition of visceral adipose tissue (VAT) promotes the malignant transformation of hormone receptor-negative mammary epithelial cells is currently unknown. We developed a novel in vitro system of malignant transformation in which non-tumorigenic human breast epithelial cells (MCF-10A) grow in soft agar when cultured with factors released from VAT. These cells, which acquire the capacity for 3D growth, show elevated aryl hydrocarbon receptor (AhR) protein and AhR target genes, suggesting that AhR activity may drive malignant transformation by VAT. AhR is a ligand-dependent transcription factor that generates biological responses to exogenous carcinogens and to the endogenous tryptophan pathway metabolite, kynurenine. The serum kynurenine to tryptophan ratio has been shown to be elevated in patients with obesity. Herein, we demonstrate that AhR inhibitors or knockdown of AhR in MCF-10A cells prevents VAT-induced malignant transformation. Specifically, VAT-induced transformation is inhibited by Kyn-101, an inhibitor for the endogenous ligand binding site of AhR. Mass spectrometry analysis demonstrates that adipocytes metabolize tryptophan and release kynurenine, which is taken up by MCF-10A cells and activates the AhR to induce CYP1B1 and promote malignant transformation. This novel hormone receptor-independent mechanism of malignant transformation suggests targeting AhR for TNBC prevention in the context of visceral adiposity.

(R,S)-trihexyphenidyl, acting via a muscarinic receptor-independent mechanism, inhibits hippocampal glutamatergic and GABAergic synaptic transmissions: Potential relevance for treatment of organophosphorus intoxication

Preclinical studies have reported that, compared to the muscarinic receptor (mAChR) antagonist atropine, (R,S)-trihexyphenidyl (THP) more effectively counters the cholinergic crisis, seizures, and neuropathology triggered by organophosphorus (OP)-induced acetylcholinesterase (AChE) inhibition. The greater effectiveness of THP was attributed to its ability to block mAChRs and N-methyl-d-aspartate-type glutamatergic receptors (NMDARs) in the brain. However, THP also inhibits α7 nicotinic receptors (nAChRs). The present study examined whether THP-induced inhibition of mAChRs, α7 nAChRs, and NMDARs is required to suppress glutamatergic synaptic transmission, whose overstimulation sustains OP-induced seizures. In primary hippocampal cultures, THP (1–30 μM) suppressed the frequency of excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) recorded from neurons in nominally Mg2+-free solution. A single sigmoidal function adequately fit the overlapping concentration-response relationships for THP-induced suppression of IPSC and EPSC frequencies yielding an IC50 of 6.3 ± 1.3 μM. Atropine (1 μM), the NMDAR antagonist d,l-2-amino-5-phosphonopentanoic acid (D,L-AP5, 50 μM), and the α7 nAChR antagonist methyllycaconitine (MLA, 10 nM) did not prevent THP-induced inhibition of synaptic transmission. THP (10 μM) did not affect the probability of transmitter release because it had no effect on the frequency of miniature IPSCs and EPSCs recorded in the presence of tetrodotoxin. Additionally, THP had no effect on the amplitudes and decay-time constants of miniature IPSCs and EPSCs; therefore, it did not affect the activity of postsynaptic GABAA and glutamate receptors. This study provides the first demonstration that THP can suppress action potential-dependent synaptic transmission via a mechanism independent of NMDAR, mAChR, and α7 nAChR inhibition.

Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy.

Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-β1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.

Phenotypic Assessment of Pathogenic Variants in GNAO1 and Response to Caffeine in C. elegans Models of the Disease.

De novo mutations affecting the G protein α o subunit (Gαo)-encoding gene (GNAO1) cause childhood-onset developmental delay, hyperkinetic movement disorders, and epilepsy. Recently, we established Caenorhabditis elegans as an informative experimental model for deciphering pathogenic mechanisms associated with GNAO1 defects and identifying new therapies. In this study, we generated two additional gene-edited strains that harbor pathogenic variants which affect residues Glu246 and Arg209-two mutational hotspots in Gαo. In line with previous findings, biallelic changes displayed a variable hypomorphic effect on Gαo-mediated signaling that led to the excessive release of neurotransmitters by different classes of neurons, which, in turn, caused hyperactive egg laying and locomotion. Of note, heterozygous variants showed a cell-specific dominant-negative behavior, which was strictly dependent on the affected residue. As with previously generated mutants (S47G and A221D), caffeine was effective in attenuating the hyperkinetic behavior of R209H and E246K animals, indicating that its efficacy is mutation-independent. Conversely, istradefylline, a selective adenosine A2A receptor antagonist, was effective in R209H animals but not in E246K worms, suggesting that caffeine acts through both adenosine receptor-dependent and receptor-independent mechanisms. Overall, our findings provide new insights into disease mechanisms and further support the potential efficacy of caffeine in controlling dyskinesia associated with pathogenic GNAO1 mutations.

Kinase Activity Is Not Required for G Protein-Coupled Receptor Kinase 4 Restraining mTOR Signaling during Cilia and Kidney Development.

G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause. G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development. To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model. Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause. These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.

Unique, Specific CART Receptor-Independent Regulatory Mechanism of CART(55-102) Peptide in Spinal Nociceptive Transmission and Its Relation to Dipeptidyl-Peptidase 4 (DDP4).

Cocaine- and amphetamine-regulated transcript (CART) peptides are involved in several physiological and pathological processes, but their mechanism of action is unrevealed due to the lack of identified receptor(s). We provided evidence for the antihyperalgesic effect of CART(55-102) by inhibiting dipeptidyl-peptidase 4 (DPP4) in astrocytes and consequently reducing neuroinflammation in the rat spinal dorsal horn in a carrageenan-evoked inflammation model. Both naturally occurring CART(55-102) and CART(62-102) peptides are present in the spinal cord. CART(55-102) is not involved in acute nociception but regulates spinal pain transmission during peripheral inflammation. While the full-length peptide with a globular motif contributes to hyperalgesia, its N-terminal inhibits this process. Although the anti-hyperalgesic effects of CART(55-102), CART(55-76), and CART(62-76) are blocked by opioid receptor antagonists in our inflammatory models, but not in neuropathic Seltzer model, none of them bind to any opioid or G-protein coupled receptors. DPP4 interacts with Toll-like receptor 4 (TLR4) signalling in spinal astrocytes and enhances the TLR4-induced expression of interleukin-6 and tumour necrosis factor alpha contributing to inflammatory pain. Depending on the state of inflammation, CART(55-102) is processed in the spinal cord, resulting in the generation of biologically active isoleucine-proline-isoleucine (IPI) tripeptide, which inhibits DPP4, leading to significantly decreased glia-derived cytokine production and hyperalgesia.

Dual roles and potential applications of exosomes in HCV infections.

The hepatitis C virus (HCV) causes severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma, which have high morbidity and mortality. Antibody targeting receptor-mediated HCV infections have limited therapeutic benefits, suggesting that the transmission of HCV infections is possibly mediated via receptor-independent mechanisms. Exosomes are membrane-enclosed vesicles with a diameter of 30-200 nm, which originate from the fusion of endosomal multivesicular bodies with the plasma membrane. Accumulating evidence suggests that exosomes have a pivotal role in HCV infections. Exosomes can transfer viral and cellular bioactive substances, including nucleic acids and proteins, to uninfected cells, thus spreading the infection by masking these materials from immunological recognition. In addition, exosomes originating from some cells can deliver antiviral molecules or prompt the immune response to inhibit HCV infection. Exosomes can be used for the diagnosis of HCV-related diseases, and are being presently evaluated as therapeutic tools for anti-HCV drug delivery. This review summarizes the current knowledge on the dual roles and potential clinical applications of exosomes in HCV infections.

Meet the First Authors.

Meet the First Authors.

Molecular networks underlying cannabinoid signaling in skeletal muscle plasticity.

The cannabinoid system is ubiquitously present and is classically considered to engage in neural and immunity processes. Yet, the role of the cannabinoid system in the whole body and tissue metabolism via central and peripheral mechanisms is increasingly recognized. The present review provides insights in (i) how cannabinoid signaling is regulated via receptor-independent and -dependent mechanisms and (ii) how these signaling cascades (might) affect skeletal muscle plasticity and physiology. Receptor-independent mechanisms include endocannabinoid metabolism to eicosanoids and the regulation of ion channels. Alternatively, endocannabinoids can act as ligands for different classic (cannabinoid receptor 1 [CB1 ], CB2 ) and/or alternative (e.g.,TRPV1, GPR55) cannabinoid receptors with a unique affinity, specificity, and intracellular signaling cascade (often tissue-specific). Antagonism of CB1 might hold clues to improve oxidative (mitochondrial) metabolism, insulin sensitivity, satellite cell growth, and muscle anabolism, whereas CB2 agonism might be a promising way to stimulate muscle metabolism and muscle cell growth. Besides, CB2 ameliorates muscle regeneration via macrophage polarization towardan anti-inflammatory phenotype, induction of MyoD and myogenin expression and antifibrotic mechanisms. Also TRPV1 and GPR55 contribute to the regulation of muscle growth and metabolism. Future studies should reveal how the cannabinoid system can be targeted to improve muscle quantity and/or quality in conditions such as ageing, disease, disuse, and metabolic dysregulation, taking into account challenges that are inherent to modulation of the cannabinoid system, such as central and peripheral side effects.