Outcomes of chronic myeloid leukemia patients after therapeutic failure to conventional tyrosine kinase inhibitors and asciminib.

Patients with chronic myeloid leukemia (CML) who have failed conventional tyrosine kinase inhibitors (cTKIs) and asciminib constitute a complex group of patients with few therapeutic options. A retrospective descriptive multicenter observational study was carried out including patients with CML who had presented a therapeutic failure to ≥ 2 cTKIs and asciminib, and had received or were not candidates to ponatinib. Of the 19 patients enrolled, 8 patients failed asciminib due to intolerance and 11 due to resistance. The most common strategy for intolerant patients was to initiate a previously administered cTKI (6/8) with dose adjustments or symptomatic management of adverse events (AEs). Of the patients who failed due to resistance, only patients who underwent progenitor transplantation achieved profound long-term responses. A frequent strategy was to use ponatinib (4/11) in patients previously considered non-candidates, with poor responses in patients in whom dose reductions were used, and severe AEs in patients at standard doses. In the remaining patients, cTKIs and other agents (interferon, hydroxyurea, citarabine, busulfan) were used with poor responses. Patients who progressed to advanced stages had a dismal prognosis. With a median follow-up of 11.2 months, overall survival of the global cohort was 73%; 100% for intolerant patients, 71% for resistant patients and 25% for those who discontinue asciminib in accelerated/blastic phase.

Digital Microscopy Augmented by Artificial Intelligence to Interpret Bone Marrow Samples for Hematological Diseases.

Analysis of bone marrow aspirates (BMAs) is an essential step in the diagnosis of hematological disorders. This analysis is usually performed based on a visual examination of samples under a conventional optical microscope, which involves a labor-intensive process, limited by clinical experience and subject to high observer variability. In this work, we present a comprehensive digital microscopy system that enables BMA analysis for cell type counting and differentiation in an efficient and objective manner. This system not only provides an accessible and simple method to digitize, store, and analyze BMA samples remotely but is also supported by an Artificial Intelligence (AI) pipeline that accelerates the differential cell counting process and reduces interobserver variability. It has been designed to integrate AI algorithms with the daily clinical routine and can be used in any regular hospital workflow.

Increased Phospho-AKT in Blood Cells from LRRK2 G2019S Mutation Carriers.

The purpose of this study was to investigate whether  differential phosphorylation states of blood markers can identify patients with LRRK2 Parkinson's disease (PD). We assessed phospho(P)-Ser-935-LRRK2 and P-Ser-473-AKT levels in peripheral blood cells from patients with G2019S LRRK2-associated PD (L2PD, n = 31), G2019S LRRK2 non-manifesting carriers (L2NMC, n = 26), idiopathic PD (iPD, n = 25), and controls (n = 40, total n = 122). We found no differences at P-Ser-935-LRRK2 between groups but detected a specific increase of P-Ser-473-AKT levels in all G2019S carriers, either L2PD or L2NMC, absent in iPD. Although insensitive to LRRK2 inhibition, our study identifies P-Ser-473-AKT as an endogenous candidate biomarker for peripheral inflammation in G2019S carriers using accessible blood cells. ANN NEUROL 2022;92:888-894.

Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search.

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

Differential Phospho-Signatures in Blood Cells Identify LRRK2 G2019S Carriers in Parkinson's Disease.

BACKGROUND: The clinicopathological phenotype of G2019S LRRK2-associated Parkinson's disease (L2PD) is similar to idiopathic Parkinson's disease (iPD), and G2019S LRRK2 nonmanifesting carriers (L2NMCs) are at increased risk for development of PD. With various therapeutic strategies in the clinical and preclinical pipeline, there is an urgent need to identify biomarkers that can aid early diagnosis and patient enrichment for ongoing and future LRRK2-targeted trials. OBJECTIVE: The objective of this work was to investigate differential protein and phospho-protein changes related to G2019S mutant LRRK2 in peripheral blood mononuclear cells from G2019S L2PD patients and G2019S L2NMCs, identify specific phospho-protein changes associated with the G2019S mutation and with disease status, and compare findings with patients with iPD. METHODS: We performed an unbiased phospho-proteomic study by isobaric label-based mass spectrometry using peripheral blood mononuclear cell group pools from a LRRK2 cohort from Spain encompassing patients with G2019S L2PD (n = 20), G2019S L2NMCs (n = 20), healthy control subjects (n = 30), patients with iPD (n = 15), patients with R1441G L2PD (n = 5), and R1441G L2NMCs (n = 3) (total N = 93). RESULTS: Comparing G2019S carriers with healthy controls, we identified phospho-protein changes associated with the G2019S mutation. Moreover, we uncovered a specific G2019S phospho-signature that changes with disease status and can discriminate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Although patients with iPD showed a differential phospho-proteomic profile, biological enrichment analyses revealed similar changes in deregulated pathways across the three groups. CONCLUSIONS: We found a differential phospho-signature associated with LRRK2 G2019S for which, consistent with disease status, the phospho-profile from PD at-risk G2019S L2NMCs was more similar to healthy controls than patients with G2019S L2PD with the manifested disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Serum MicroRNAs Predict Isolated Rapid Eye Movement Sleep Behavior Disorder and Lewy Body Diseases.

BACKGROUND: Isolated rapid eye movement sleep behavior disorder (IRBD) is a well-established clinical risk factor for Lewy body diseases (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). OBJECTIVE: To elucidate whether serum microRNA (miRNA) deregulation in IRBD can antedate the diagnosis of LBD by performing a longitudinal study in different progression stages of IRBD before and after LBD diagnosis and assessing the predictive performance of differentially expressed miRNAs by machine learning-based modeling. METHODS: Using genome-wide miRNA analysis and real-time quantitative polymerase chain reaction validation, we assessed serum miRNA profiles from patients with IRBD stratified by dopamine transporter (DaT) single-photon emission computed tomography into DaT-negative IRBD (n = 17) and DaT-positive IRBD (n = 21), IRBD phenoconverted into LBD (n = 13), and controls (n = 20). Longitudinally, we followed up the IRBD cohort by studying three time point serum samples over 26 months. RESULTS: We found sustained cross-sectional and longitudinal deregulation of 12 miRNAs across the RBD continuum, including DaT-negative IRBD, DaT-positive IRBD, and LBD phenoconverted IRBD (let-7c-5p, miR-19b-3p, miR-140, miR-22-3p, miR-221-3p, miR-24-3p, miR-25-3p, miR-29c-3p, miR-361-5p, miR-425-5p, miR-4505, and miR-451a) (false discovery rate P < 0.05). Age- and sex-adjusted predictive modeling based on the 12 differentially expressed miRNA biosignatures discriminated IRBD and PD or DLB from controls with an area under the curve of 98% (95% confidence interval: 89-99%). CONCLUSIONS: Besides clinical diagnosis of IRBD or imaging markers such as DaT single-photon emission computed tomography, specific miRNA biosignatures alone hold promise as progression biomarkers for patients with IRBD for predicting PD and DLB clinical outcomes. Further miRNA studies in other PD at-risk populations, such as LRRK2 mutation asymptomatic carriers or hyposmic subjects, are warranted. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Pharmacological and behavioral divergence of ketamine enantiomers: implications for abuse liability.

Ketamine, a racemic mixture of (S)-ketamine and (R)-ketamine enantiomers, has been used as an anesthetic, analgesic and more recently, as an antidepressant. However, ketamine has known abuse liability (the tendency of a drug to be used in non-medical situations due to its psychoactive effects), which raises concerns for its therapeutic use. (S)-ketamine was recently approved by the United States' FDA for treatment-resistant depression. Recent studies showed that (R)-ketamine has greater efficacy than (S)-ketamine in preclinical models of depression, but its clinical antidepressant efficacy has not been established. The behavioral effects of racemic ketamine have been studied extensively in preclinical models predictive of abuse liability in humans (self-administration and conditioned place preference [CPP]). In contrast, the behavioral effects of each enantiomer in these models are unknown. We show here that in the intravenous drug self-administration model, the gold standard procedure to assess potential abuse liability of drugs in humans, rats self-administered (S)-ketamine but not (R)-ketamine. Subanesthetic, antidepressant-like doses of (S)-ketamine, but not of (R)-ketamine, induced locomotor activity (in an opioid receptor-dependent manner), induced psychomotor sensitization, induced CPP in mice, and selectively increased metabolic activity and dopamine tone in medial prefrontal cortex (mPFC) of rats. Pharmacological screening across thousands of human proteins and at biological targets known to interact with ketamine yielded divergent binding and functional enantiomer profiles, including selective mu and kappa opioid receptor activation by (S)-ketamine in mPFC. Our results demonstrate divergence in the pharmacological, functional, and behavioral effects of ketamine enantiomers, and suggest that racemic ketamine's abuse liability in humans is primarily due to the pharmacological effects of its (S)-enantiomer.

Pharmacological Characterization of the Imipridone Anticancer Drug ONC201 Reveals a Negative Allosteric Mechanism of Action at the D2 Dopamine Receptor.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated ß-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

Heteromerization between α2A adrenoceptors and different polymorphic variants of the dopamine D4 receptor determines pharmacological and functional differences. Implications for impulsive-control disorders.

Polymorphic alleles of the human dopamine D4 receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D4.7 receptor variant and attention deficit hyperactivity disorder (ADHD). The α2A adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α2A adrenoceptor (α2AR), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D4 receptor (D4R) and α2AR in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D4R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α2AR forms functional heteromers with D4R and weather these heteromers show different properties depending on the D4R variant involved. Using cortical brain slices from hD4.7R knock-in and wild-type mice, here, we demonstrate that α2AR and D4R heteromerize and constitute a significant functional population of cortical α2AR and D4R. Moreover, in cortical slices from wild-type mice and in cells transfected with α2AR and D4.4R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD4.7R knock-in mice and in cells expressing the D4.7R polymorphic variant. We also show a lack of efficacy of D4R ligands to promote G protein activation and signaling only within the α2AR-D4.7R heteromer. Taken together, our results suggest that α2AR-D4R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy.

Inflammatory dysregulation in women with an eating disorder: Relationships with altered emotional reactivity.

BACKGROUND: Some studies suggest that inflammatory signaling dysregulation may contribute to eating disorder (ED) pathophysiology. However, little is known about the influence of inflammatory response on altered processes seen among patients with ED, such as emotional processing and reactivity. OBJECTIVES: The objectives were: (a) to investigate the systemic inflammatory response in ED women; and (b) to analyze the role of inflammatory markers in emotional reactivity. METHOD: Concentrations of several intercellular and intracellular inflammatory mediators (cytokines, prostaglandin by-products and enzymes, TBARS, and MAPK proteins) were quantified in plasma and PBMCs from 68 women with an ED (m = 22.01 years, SD = 9.15) and 35 healthy controls (m = 18.54 years, SD = 4.21). Moreover, emotional reactivity to affective pictures (those without either food or thinness content) was studied using the adult (>18 years old) sample (n = 41). RESULTS: Between-group differences were revealed for most markers (TNF-α, PGE2 , COX2, and ratio of activated MAPK proteins), pointing to increased inflammatory response in patients (p < .01). Women with ED showed heightened emotional reactivity, regardless of picture valence. Principal components derived from inflammatory markers showed an explanatory loading on patient's emotional reaction, in terms of valence and arousal. CONCLUSION: This study corroborates the altered systemic inflammatory response in patients with ED. The inflammatory dysregulation may contribute to ED phenotype, as seen by its relationship with heightened emotional reactivity, even though the inflammatory markers were not evaluated throughout the emotional reactivity protocol.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

MicroRNA Deregulation in Blood Serum Identifies Multiple System Atrophy Altered Pathways.

BACKGROUND AND OBJECTIVES: MicroRNA (miRNA) changes are observed in PD but remain poorly explored in other α-synucleinopathies such as MSA. METHODS: By genome-wide analysis we profiled microRNA expression in serum from 20 MSA cases compared to 40 controls. By qPCR we validated top differentially expressed microRNAs in another sample of 20 MSA and 20 controls. We also assessed the expression of MSA differentially expressed microRNAs in two consecutive sets of 19 and 18 PD patients. RESULTS: In the discovery set we identified 25 differentially expressed microRNAs associated with MSA, which are related to prion disease, fatty acid metabolism, and Notch signaling. Among these, we selected nine differentially expressed microRNAs and by qPCR confirmed array findings in a second MSA sample. MicroRNA-7641 and microRNA-191 consistently differentiated between MSA and PD. CONCLUSIONS: Serum microRNA changes occur in MSA and may reflect disease-associated mechanisms. We identified two microRNAs which may differentiate MSA from PD. © 2020 International Parkinson and Movement Disorder Society.

Identification of Positive Allosteric Modulators of the D1 Dopamine Receptor That Act at Diverse Binding Sites.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and ß-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both ß-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist.

The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and ß arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays.

Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer.

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of ß-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted ß-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists.

Differential serum microRNAs in premotor LRRK2 G2019S carriers from Parkinson's disease.

The LRRK2 G2019S pathogenic mutation causes LRRK2-associated Parkinson's disease (L2PD) with incomplete penetrance. LRRK2 non-manifesting carriers (L2NMC) are at PD high risk but predicting pheno-conversion is challenging given the lack of progression biomarkers. To investigate novel biomarkers for PD premotor stages, we performed a longitudinal microRNA (miRNA) assessment of serum samples from G2019S L2NMC followed-up over 8 years. Our cohort consisted of G2019S L2NMC stratified by dopamine transporter single-photon emission computed tomography (DaT-SPECT) into DaT-negative (n = 20) and DaT-positive L2NMC (n = 20), pheno-converted G2019S L2PD patients (n = 20), idiopathic PD (iPD) (n = 19), and controls (n = 40). We also screened a second cohort of L2PD patients (n = 19) and controls (n = 20) (Total n = 158). Compared to healthy controls, we identified eight deregulated miRNAs in DaT-negative L2NMC, six in DaT-positive L2NMC, and one in L2PD. Between groups, the highest miRNA differences, 24 candidate miRNAs, occurred between DaT-positive L2NMC and L2PD. Longitudinally, we found 11 common miRNAs with sustained variation in DaT-negative and DaT-positive L2NMCs compared to their baselines. Our study identifies novel miRNA alterations in premotor stages of PD co-occurring with progressive DaT-SPECT decline before motor manifestation, whose deregulation seems to attenuate after the diagnosis of L2PD. Moreover, we identified four miRNAs with relatively high discriminative ability (AUC = 0.82) between non-pheno-converted DaT-positive G2019S carriers and pheno-converted L2PD patients (miR-4505, miR-8069, miR-6125, and miR-451a), which hold potential as early progression biomarkers for PD.

G Protein-Coupled Receptor Kinase 2 Selectively Enhances ß-Arrestin Recruitment to the D2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation.

The D2 dopamine receptor (D2R) signals through both G proteins and ß-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. ß-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for ß-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for ß-arrestin recruitment to the D2R, and the role of GRKs in D2R-ß-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which ß-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated ß-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased ß-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-ß-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance ß-arrestin recruitment and activation independently of receptor phosphorylation.

Dysfunction of Inflammatory Pathways and Their Relationship With Psychological Factors in Adult Female Patients With Eating Disorders.

The attempts to clarify the origin of eating disorders (ED) have not been completely successful and their etiopathogenesis remains unknown. Current research shows an activation of the immune response in neuropsychiatric diseases, including ED. We aimed to investigate immune response parameters in patients with ED and to identify psychological factors influencing the inflammatory response. The relationship between inflammation markers and impulsivity and affective symptomatology was explored as well. Thirty-four adult female patients with current diagnosis of ED, none of them under psychopharmacological treatment (excluding benzodiazepines), were included in this study. Patients were compared with a healthy control group of fifteen adult females. The levels of inflammatory markers and indicators of oxidative/nitrosative stress were evaluated in plasma and/or in peripheral blood mononuclear cells (PBMCs). Subjects were assessed by means of different ED evaluation tools. Additionally, the Barratt Impulsiveness Scale, the Montgomery-Asberg Depression Rating Scale and the Hamilton Anxiety Rating Scale were also employed. Patients with ED shown increased plasma levels of the pro-inflammatory nuclear factor kappa B (NFκB) and the cytokine tumor necrosis factor-alpha (TNF-α), among other factors and an increment in the oxidative/nitrosative stress as well as increased glucocorticoid receptor (GR) expression levels in their PBMCs. Moreover, the inflammatory prostaglandin E2 (PGE2) correlated with impulsiveness and the anti-inflammatory prostaglandin J2 (15d-PGJ2) correlated with depressive symptomatology. Our results point towards a relationship between the immune response and impulsiveness and between the immune response and depressive symptomatology in female adult patients with ED.