Effects of combined prenatal exposure to air pollution and maternal stress on immune and dopaminergic gene expression in the gut-brain axis

Abstract BACKGROUND Pediatric brain tumors are leading cause of cancer-related deaths in children. ONC201, a selective dopamine receptor D2 (DRD2) antagonist and ClpP agonist, is in phase I-III clinical trials for various cancers, notably H3K27 mutant gliomas. However, resistance has been reported and its mechanism remains unclear. This study was to assess therapeutic efficacy and adaptive drug responses of ONC201 longitudinally across multiple patient -derived orthotopic mouse models (PDOX). Additionally, two patient samples with ONC201 treatment failure were obtained post-mortem. METHODS DRD2 mRNA were profiled by RNA-seq. ONC201 cytotoxicity was evaluated in vitro in PDOX-derived tumor cells. Total of 11 PDOX, 6 glioblastoma (GBM), 3 medulloblastoma (MB), and 2 ependymoma (EPN) were treated with ONC201 (125mg/kg) 2-3 weeks after tumor implantation and monitored for survival times. Immunohistochemistry was used to examine phenotypical changes. Single-cell RNA sequencing was performed to uncover the trajectory of drug responsiveness during the course of ONC 201 treatment and identify treatment-responsive and resistant cell subpopulations. RESULTS DRD2 expression varied across tumors with highest in MBs (RNA seq FPKM=4), moderate in GBMs (FPKM=1.5-2.0), and low in EPNs (FPKM=0.4-0.5). ONC201 inhibited tumor cell proliferation in 8 of 11 models in vitro (IC50 = 0.26–0.52 µM) and significantly extended animal survival by 10–21% (P < 0.05) in 50% (3/6) GBMs whereas no benefits in MB or EPNs. Single-cell analysis revealed the trajectory of drug responses, identified drug-responsive and -resistant cell populations and potential resistance-associated genes. CONCLUSION DRD2 expression and in vitro cytotoxicity did not reliably predict in vivo efficacy. ONC201 demonstrated anti-tumor efficacy in pediatric GBMs, extending beyond H3K27 mutant tumors, but resistance emerged with enriched population of therapy-resistant cells. These findings highlight Inclusion criteria other than DRD2 expression is needed for ONC201 therapy and suggest combination therapies to prevent tumor recurrence.

Inverse salt sensitivity, an increase in blood pressure (BP) when sodium intake is reduced, affects about 10-15% of the population, yet the mechanisms underlying this alteration in BP are not well understood. The renal dopamine D2 receptor (D2R) plays a critical role in maintaining normal BP and preventing inflammation and tissue injury. The DRD2 is highly polymorphic, and single nucleotide polymorphisms (SNPs) in this gene impair DRD2 synthesis and stability. Specifically, rs6277 SNP in exon 7 of DRD2 is associated with decreased D2R expression and is present in some individuals with hypertension. We have reported that human renal proximal tubular cells with this SNP have decreased D2R mRNA and protein expressions and increased renal Na+ pump/transporter expression. To study the effects of rs6277 on sodium balance and BP, using CRISPR-Cas9, we generated C57Bl/6 mice lacking their own Drd2 but instead express either the human DRD2 wild-type ( DRD2 WT) or rs6277 ( DRD2 Mut). Male and female mice were placed for one week on three distinct salt diets: normal salt (NS; 0.4% NaCl), high salt (HS; 4% NaCl), and low salt (LS; less than 0.08% NaCl) diets. On NS diet, BPs (measured by tail-cuff plethysmography under pentobarbital anesthesia) were slightly higher in male DRD2 Mut than DRD2 WT mice (79±3 vs 73±±0.5 mm Hg; P<0.04; n=5-7/group) while BPs were similar in female DRD2 Mut and DRD2 WT mice (77±3 vs 75±3 mm Hg: n=8/group). On HS diet, BPs were similar in DRD2 WT and DRD2 Mut mice (males 89±2 vs 92±3; females 79±3 vs 87±5 mm Hg). However, on LS diet DRD2 Mut had higher BPs than DRD2 WT mice (males: 72±2 vs 90±2 P<0.001; females: 68±1 vs 88±3 mm Hg, P<0.001). Thus, in DRD2 WT mice, BP increased on HS diet and decreased on LS, while in DRD2 Mut mice, BP increased on both LS and HS diets. There were no significant differences in urinary sodium excretion between DRD2 WT and DRD2 Mut male and female mice on the different diets. These findings suggest that alterations in DRD2 expression/function may be the underlying cause of inverse salt sensitivity of BP because the presence of DRD2 rs6277 is associated with inverse salt sensitivity in mice and humans. Moreover, the increased BP in DRD2 rs6277 mice on LS is independent of urinary sodium excretion.

Swiss-Webster and C57BL/6 mice are differentially sensitive to the stimulant effects of methamphetamine.

A result of the ongoing opioid epidemic has been a significant rise in the rates of opioid use during pregnancy. This includes use of maintenance medications for opioid use disorder (MOUDs), such as methadone, which are the standard of care for pregnant people with an opioid use disorder (OUD). Although the use of MOUDs leads to better neonatal outcomes in exposed offspring compared to those born from individuals with untreated OUD, the pharmacology of MOUDs is similar to misused opioids. Despite the high prevalence of prenatal exposure to opioids, including MOUDs, our understanding of the long-term consequences of these exposures in offspring is limited. Prenatal drug exposure is known to be a risk factor for future substance use disorder and mood disorders, yet, how prenatal opioid exposure influences ethanol intake in adult offspring and associated affective behaviors has not been examined. Using a rat model of prenatal methadone exposure (PME), which included twice daily methadone injections from gestational day 3-20, this study assessed ethanol intake in adult offspring and how exposure to forced swim stress (FSS) altered ethanol intake, in addition to examination of depressive-like behavior during the FSS. Given the role of the basolateral amygdala (BLA) in emotion and reward processing, we also conducted patch clamp electrophysiology experiments from BLA neurons to investigate changes in synaptic transmission and gene expression of neuromodulatory systems that are known to influence emotion and reward processing. Females with a history of PME consumed less ethanol than control females, with no effects of PME on ethanol intake evident in males. While PME increased immobility during FSS in both males and females, FSS had no effects on ethanol intake. PME increased glutamate transmission and altered dopamine D1, D2, and D3 receptor and mu opioid receptor mRNA in the BLA of females, but not in males. Collectively, this study identified impairments in emotion and reward processing, in addition to alterations in synaptic function and gene expression in the BLA of females with a history of PME, supporting previous findings from our lab demonstrating that female offspring are more sensitive to the long-term effects of PME.

Correlation of repetitive behaviors in deer mice with striatal mRNA expression of endogenous opioids and mu, delta, kappa, and dopamine receptors: A preliminary report.

Impulsivity is often considered a risk factor for drug addiction; however, not all evidence supports this view. In the present study, we used a food reward delay-discounting task (DDT) to categorize rats as low-, middle-, and high-impulsive but failed to find any difference among these groups in the acquisition and maintenance of cocaine self-administration (SA), regardless of electrical footshock punishment. Additionally, there were no group differences in locomotor responses to acute cocaine in rats with or without a history of cocaine SA. Unexpectedly, chronic cocaine SA selectively increased impulsive choice in low-impulsive rats. Resting-state fMRI analysis revealed a positive correlation between impulsivity and cerebral blood volume in the midbrain, thalamus, and auditory cortex. Using these three regions as seeds, we observed a negative correlation between impulsivity and functional connectivity between the midbrain and frontal cortex, as well as between the thalamus and frontal cortex (including the orbitofrontal, primary, and parietal cortices) in low-impulsive rats. These correlations were attenuated following chronic cocaine SA. RNAscope in situ hybridization assays revealed a significant reduction in dopamine (DA) D1, D2, and D3 receptor mRNA expression in the corticostriatal regions of low-impulsive rats after cocaine SA. Our findings challenge the widely held view that impulsivity is a vulnerability factor for cocaine use disorder. Instead, chronic cocaine use appears to selectively increase impulsive choice decision-making in normally low-impulsive rats, associated with reduced functional connectivity and DA receptor expression in the mesocorticolimbic DA network.

The use of psilocybin to treat alcohol use disorder is very promising, but its mechanisms of action remain poorly understood. We combined behavioural, pharmacological and gene expression analyses to decipher the mechanisms of action of psilocybin, for the first time, when injected into the brain. Male Long Evans rats underwent chronic operant ethanol self-administration before testing the effect of intraperitoneal psilocybin or directly within the nucleus accumbens core or the ventral tegmental area. Transcripts from the dopaminergic system were quantified in the nucleus accumbens and prefrontal cortex. Psilocybin significantly reduced (by 50%) ethanol self-administration when injected 4 h before the session either intraperitoneally (1 mg/kg) or directly within the left nucleus accumbens (0.15 μg) but not the right nucleus accumbens or the left ventral tegmental area. The effect of intraperitoneal injection of psilocybin was prevented by intra-left nucleus accumbens injection of 0.3 μg of the 5-HT2A receptor antagonist ketanserin. In rats that self-administered ethanol but not in those self-administering saccharin, dopamine D2 receptor (D2R) mRNA was increased in both the nucleus accumbens and the prefrontal cortex by psilocybin, while dopamine D1 receptor mRNA was increased only in the prefrontal cortex. As in humans, psilocybin reduced ethanol self-administration in rats through the 5-HT2A receptor within the left nucleus accumbens, possibly through increased D2R expression. Our results open unexpected perspectives regarding the hemispheric lateralization of psychedelic effects.

Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.

Regulation of plasma glucose levels by central dopamine D2 receptors is impaired in type 1 but not type 2 diabetic mouse models

Autism Spectrum Disorder (ASD) is a progressive neurodevelopmental disorder mainly characterized by deficits in social communication and stereotyped behaviors and interests. Here, we aimed to investigate the state of several key players in the dopamine and glutamate neurotransmission systems in the valproic acid (VPA) animal model that was administered to E12.5 pregnant females as a single dose (450 mg/kg). We report no alterations in the number of mesencephalic dopamine neurons or in protein levels of tyrosine hydroxylase in either the striatum or the nucleus accumbens. In females prenatally exposed to VPA, levels of dopamine were slightly decreased while the ratio of DOPAC/dopamine was increased in the dorsal striatum, suggesting increased turn-over of dopamine tone. In turn, levels of D1 and D2 dopamine receptor mRNAs were increased in the nucleus accumbens of VPA mice suggesting upregulation of the corresponding receptors. We also report decreased protein levels of striatal parvalbumin and increased levels of p-mTOR in the cerebellum and the motor cortex of VPA mice. mRNA levels of mGluR1, mGluR4, and mGluR5 and the glutamate receptor subunits NR1, NR2A, and NR2B were not altered by VPA, nor were protein levels of NR1, NR2A, and NR2B and those of BDNF and TrkB. These findings are of interest as clinical trials aiming at the dopamine and glutamate systems are being considered.

ObjectiveMost men suffering from depression have different degrees of erectile dysfunction (ED), but the relationship between depression and ED is not clear. This study explored the effect of depression on erectile function in rats and the underlying mechanism.MethodsThe potential targets and key signaling pathways of depression and ED were predicted through bioinformatics analysis, and a depression rat model was established by inducing chronic restraint stress. Pathological changes in rat penis tissue were studied by hematoxylin and eosin staining. The serum dopamine level was quantified by an enzyme-linked immunosorbent assay. The expression of related proteins and mRNA was detected by western blotting and real-time quantitative reverse transcription-polymerase chain reaction.ResultsHematoxylin and eosin staining showed pathological damage in the penile tissue of the model group rats. The serum dopamine level, dopamine receptor D2 (DRD2) and solute carrier family 6 member 3 (SLC6A3) protein levels in penile tissue, and DRD2 and SLC6A3 mRNA levels were lower in the model group than in the control group.ConclusionThe decrease in erectile function in the depression rat model was related to dysfunction of the dopamine system and dopaminergic synapse signaling pathway.

Small intestine neuromuscular dysfunction in a mouse model of dextran sulfate sodium-induced ileitis: Involvement of dopaminergic neurotransmission

When the rat D3 dopamine receptor (D3R) was cloned and the distribution of its mRNA examined in 1990-1991, it attracted attention due to its peculiar distribution in the brain quite different from that of its closest relative, the D2 receptor. In the rat brain, the D3R mRNA is enriched in the limbic striatum as opposed to the D2 receptor, which is highly expressed in the motor striatal areas. Later studies in the primate and human brain confirmed relative enrichment of the D3R in the limbic striatum but also demonstrated higher abundance of theD3R in the primate as compared to the rodent brain. Additionally, in the rodent brain, the D3R in the dorsal striatum appears to be co-expressed with the D1 dopamine receptor-bearing striatal neurons giving rise to the direct output striatal pathway, although the picture is less clear with respect to the nucleus accumbens. In contrast, in the primate striatum, the D3R co-localizes with the D2 receptor throughout the basal ganglia as well as in extrastriatal brain areas. The relative abundance of the D3R in the limbic striatum, its output structures, secondary targets, and some of the other connected limbic territories may underpin its role in reward, drug dependence, and impulse control. Selective expression of D3R in the brain proliferative areas may point to its important role in the neural development as well as in neurodevelopmental abnormalities associated with schizophrenia and other developmental brain disorders.

An increasing body of evidence suggests that impaired synapse development and function are associated with schizophrenia; however, the underlying molecular pathophysiological mechanism of the disease remains largely unclear. We conducted a family-based study combined with molecular and cellular analysis using induced pluripotent stem cell (iPSC) technology. We generated iPSCs from patients with familial schizophrenia, differentiated these cells into neurons, and investigated the molecular and cellular phenotypes of the patient’s neurons. We identified multiple altered synaptic functions, including increased glutamatergic synaptic transmission, higher synaptic density, and altered splicing of dopamine D2 receptor mRNA in iPSC-derived neurons from patients. We also identified patients’ specific genetic mutations using whole-exome sequencing. Our findings support the notion that altered synaptic function may underlie the molecular and cellular pathophysiology of schizophrenia, and that multiple genetic factors cooperatively contribute to the development of schizophrenia.

Maternal immune activation leads to atypical turning asymmetry and reduced DRD2 mRNA expression in a rat model of schizophrenia

Caffeine – treat or trigger? Disparate behavioral and long-term dopaminergic changes in control and schizophrenia-like Wisket rats

BackgroundONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer.MethodsCell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1fl/flp53fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer.ResultsIncreasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1fl/fl/p53fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice.ConclusionONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.

ObjectiveDopamine D2‐like receptors – mainly dopamine D2 receptors (D2R) and dopamine D3 receptors (D3R) – are believed to be greatly involved in the pathology of Parkinson disease (PD) progression. However, these receptors have not been precisely examined in PD patients. Our aim was to quantitatively calculate the exact densities of dopamine D1 receptors (D1R), D2R, and D3R in control, Alzheimer disease (AD), and Lewy body disease (LBD) patients (including PD, Dementia with Lewy bodies, and Parkinson disease dementia); and analyze the relationship between dopamine receptors and clinical PD manifestations.MethodsWe analyzed the densities of D1R, D2R, and D3R in the striatum and substantia nigra (SN) using a novel quantitative autoradiography procedure previously developed by our group. We also examined the expression of D2R and D3R mRNA in the striatum by in situ hybridization.ResultsThe results showed that although no differences of striatal D1R were found among all groups; D2R was significantly decreased in the striatum of PD patients when compared with control and AD patients. Some clinical manifestations: age of onset, PD stage, dopamine responsiveness, and survival time after onset; showed a better correlation with striatal D1R + D3R densities combined compared to D1R or D3R alone.InterpretationThere is a possibility that we may infer the results in diagnosis, treatment, and prognosis of PD by detecting D1R + D3R as opposed to using dopamine D1 or D3 receptors alone. This is especially true for elderly patients with low D2R expression as is common in this disease.

Time-course of changes in key catecholaminergic receptors and trophic systems in rat brain after antidepressant administration