Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors.

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and âˆ¼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.

Integrating TSPO PET imaging and transcriptomics to unveil the role of neuroinflammation and amyloid-ß deposition in Alzheimer's disease.

PURPOSE: Despite the revealed role of immunological dysfunctions in the development and progression of Alzheimer's disease (AD) through animal and postmortem investigations, direct evidence regarding the impact of genetic factors on microglia response and amyloid-ß (Aß) deposition in AD individuals is lacking. This study aims to elucidate this mechanism by integrating transcriptomics and TSPO, Aß PET imaging in clinical AD cohort. METHODS: We analyzed 85 patients with PET/MR imaging for microglial activation (TSPO, [18F]DPA-714) and Aß ([18F]AV-45) within the prospective Alzheimer's Disease Immunization and Microbiota Initiative Study Cohort (ADIMIC). Immune-related differentially expressed genes (IREDGs), identified based on AlzData, were screened and verified using blood samples from ADIMIC. Correlation and mediation analyses were applied to investigate the relationships between immune-related genes expression, TSPO and Aß PET imaging. RESULTS: TSPO uptake increased significantly both in aMCI (P < 0.05) and AD participants (P < 0.01) and showed a positive correlation with Aß deposition (r = 0.42, P < 0.001). Decreased expression of TGFBR3, FABP3, CXCR4 and CD200 was observed in AD group. CD200 expression was significantly negatively associated with TSPO PET uptake (r =-0.33, P = 0.013). Mediation analysis indicated that CD200 acted as a significant mediator between TSPO uptake and Aß deposition (total effect B = 1.92, P = 0.004) and MMSE score (total effect B =-54.01, P = 0.003). CONCLUSION: By integrating transcriptomics and TSPO PET imaging in the same clinical AD cohort, this study revealed CD200 played an important role in regulating neuroinflammation, Aß deposition and cognitive dysfunction.

Seasonal expressions of the translocator protein (18 kDa), voltage-dependent anion channel, and steroidogenic acute regulatory protein in the scent glands of muskrats (Ondatra zibethicus).

Steroidogenesis machinery involves the steroidogenic acute regulatory protein (StAR), which regulates cholesterol transfer within the mitochondria, and the transport of cholesterol via a channel composed of 18-kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC) plus some accessory proteins. In this study, we investigated the immunolocalizations and expressions of StAR, TSPO, VDAC and cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. StAR, TSPO, VDAC and CYP11A1 were immunolocalized in the scent glandular, interstitial and epithelial cells in both breeding and non-breeding seasons with stronger immunostaining in the breeding season. The mRNA expression levels of StAR, TSPO, VDAC and CYP11A1 were higher in the scent glands of the breeding season than those of the non-breeding season. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) as well as scent glandular T and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to the lipid metabolic process, integral component of membrane, and steroid hormone receptor activity and hormone activity using GO analysis. Further in vitro study verified that StAR, TSPO, VDAC and CYP11A1 expression levels increased significantly after human chorionic gonadotropin, hCG/FSH treatment compared with the control group. The KEGG pathway enriched by differentially expressed genes detected to be involved in endocrine system or amino acid metabolism. These findings suggested that the scent glands of the muskrats have ability to synthesize steroids de novo, and that the steroid hormones may have an important regulatory role in the scent glandular function via an autocrine/paracrine pathway.

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages.

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

Prognostic Value of TSPO PET Before Radiotherapy in Newly Diagnosed IDH-Wild-Type Glioblastoma.

The 18-kDa translocator protein (TSPO) is gaining recognition as a relevant target in glioblastoma imaging. However, data on the potential prognostic value of TSPO PET imaging in glioblastoma are lacking. Therefore, we investigated the association of TSPO PET imaging results with survival outcome in a homogeneous cohort of glioblastoma patients. Methods: Patients were included who had newly diagnosed, histologically confirmed isocitrate dehydrogenase (IDH)-wild-type glioblastoma with available TSPO PET before either normofractionated radiotherapy combined with temozolomide or hypofractionated radiotherapy. SUVmax on TSPO PET, TSPO binding affinity status, tumor volumes on MRI, and further clinical data, such as O 6-alkylguanine DNA methyltransferase (MGMT) and telomerase reverse transcriptase (TERT) gene promoter mutation status, were correlated with patient survival. Results: Forty-five patients (median age, 63.3 y) were included. Median SUVmax was 2.2 (range, 1.0-4.7). A TSPO PET signal was associated with survival: High uptake intensity (SUVmax > 2.2) was related to significantly shorter overall survival (OS; 8.3 vs. 17.8 mo, P = 0.037). Besides SUVmax, prognostic factors for OS were age (P = 0.046), MGMT promoter methylation status (P = 0.032), and T2-weighted MRI volume (P = 0.031). In the multivariate survival analysis, SUVmax in TSPO PET remained an independent prognostic factor for OS (P = 0.023), with a hazard ratio of 2.212 (95% CI, 1.115-4.386) for death in cases with a high TSPO PET signal (SUVmax > 2.2). Conclusion: A high TSPO PET signal before radiotherapy is associated with significantly shorter survival in patients with newly diagnosed IDH-wild-type glioblastoma. TSPO PET seems to add prognostic insights beyond established clinical parameters and might serve as an informative tool as clinicians make survival predictions for patients with glioblastoma.

Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution.

The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. Here we show that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. We used in vivo genome editing in Drosophila melanogaster to evaluate the fitness effects of each putative resistance mutation and found that pleiotropy both facilitates and constrains the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.

Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.

In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.

G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner.

Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). In this study, we used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1ß2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, our findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.

External amino acid residues of insect GABA receptor channels dictate the action of the isoxazoline ectoparasiticide fluralaner.

BACKGROUND: Fluralaner is the first isoxazoline ectoparasiticide developed to protect companion animals from fleas and ticks. Fluralaner primarily inhibits arthropod γ-aminobutyric acid receptors (GABARs), which are ligand-gated ion channels comprising five subunits arranged around the channel pore. We previously reported that the action site of fluralaner resides at the M1-M3 transmembrane interface between adjacent GABAR subunits. To investigate whether fluralaner interacts with the second transmembrane segment (M2) located deep in the interface, we generated four housefly RDL GABAR mutants with non-conservative amino acid substitutions in the M2 region. RESULTS: Electrophysiological analysis of GABARs expressed in Xenopus oocytes indicated that S313A and S314A mutants exhibited fluralaner sensitivities similar to that of the wild type. M312S mutant exhibited approximately seven-fold lower sensitivity than that of the wild type. Notably, the N316L mutant was almost insensitive to fluralaner. CONCLUSION: The findings of this study indicate that the conserved external amino acid residues of insect GABAR channels play a critical role in the antagonistic effect of fluralaner. © 2023 Society of Chemical Industry.

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma.

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance.

APOE ε4 gene dose effect on imaging and blood biomarkers of neuroinflammation and beta-amyloid in cognitively unimpaired elderly.

BACKGROUND: Neuroinflammation, characterized by increased reactivity of microglia and astrocytes in the brain, is known to be present at various stages of the Alzheimer's disease (AD) continuum. However, its presence and relationship with amyloid pathology in cognitively normal at-risk individuals is less clear. Here, we used positron emission tomography (PET) and blood biomarker measurements to examine differences in neuroinflammation and beta-amyloid (Aß) and their association in cognitively unimpaired homozygotes, heterozygotes, or non-carriers of the APOE ε4 allele, the strongest genetic risk for sporadic AD. METHODS: Sixty 60-75-year-old APOE ε4 homozygotes (n = 19), heterozygotes (n = 21), and non-carriers (n = 20) were recruited in collaboration with the local Auria biobank. The participants underwent 11C-PK11195 PET (targeting 18-kDa translocator protein, TSPO), 11C-PiB PET (targeting Aß), brain MRI, and neuropsychological testing including a preclinical cognitive composite (APCC). 11C-PK11195 distribution volume ratios and 11C-PiB standardized uptake value ratios (SUVRs) were calculated for regions typical for early Aß accumulation in AD. Blood samples were drawn for measuring plasma glial fibrillary acidic protein (GFAP) and plasma Aß1-42/1.40. RESULTS: In our cognitively unimpaired sample, cortical 11C-PiB-binding increased according to APOE ε4 gene dose (median composite SUVR 1.47 (range 1.38-1.66) in non-carriers, 1.55 (1.43-2.02) in heterozygotes, and 2.13 (1.61-2.83) in homozygotes, P = 0.002). In contrast, cortical composite 11C-PK11195-binding did not differ between the APOE ε4 gene doses (P = 0.27) or between Aß-positive and Aß-negative individuals (P = 0.81) and associated with higher Aß burden only in APOE ε4 homozygotes (Rho = 0.47, P = 0.043). Plasma GFAP concentration correlated with cortical 11C-PiB (Rho = 0.35, P = 0.040), but not 11C-PK11195-binding (Rho = 0.13, P = 0.47) in Aß-positive individuals. In the total cognitively unimpaired population, both higher composite 11C-PK11195-binding and plasma GFAP were associated with lower hippocampal volume, whereas elevated 11C-PiB-binding was associated with lower APCC scores. CONCLUSIONS: Only Aß burden measured by PET, but not markers of neuroinflammation, differed among cognitively unimpaired elderly with different APOE ε4 gene dose. However, APOE ε4 gene dose seemed to modulate the association between neuroinflammation and Aß.

Cytosine methylation in GABA B1 receptor identifies alcohol-related changes for men in blood and brain tissues.

AIMS: Alcohol use alters the reward signaling processes contributing to the development of addiction. We studied the effects of alcohol use disorder (AUD) on brain regions and blood of deceased women and men to examine sex-dependent differences in epigenetic changes associated with AUD. We investigated the effects of alcohol use on the gene promoter methylation of GABBR1 coding for GABAB receptor subunit 1 in blood and brain. METHODS: We chose six brain regions associated with addiction and the reward pathway (nucleus arcuatus, nucleus accumbens, the mamillary bodies, amygdala, hippocampus and anterior temporal cortex) and performed epigenetic profiling of the proximal promoter of the GABBR1 gene of post-mortem brain and blood samples of 17 individuals with AUD pathology (4 female, 13 male) and 31 healthy controls (10 female, 21 male). RESULTS: Our results show sex-specific effects of AUD on GABBR1 promoter methylation. Especially, CpG -4 showed significant tissue-independent changes and significantly decreased methylation levels for the AUD group in the amygdala and the mammillary bodies of men. We saw prominent and consistent change in CpG-4 across all investigated tissues. For women, no significant loci were observed. CONCLUSION: We found sex-dependent differences in GABBR1 promoter methylation in relation to AUD. CpG-4 hypomethylation in male individuals with AUD is consistent for most brain regions. Blood shows similar results without reaching significance, potentially serving as a peripheral marker for addiction-associated neuronal adaptations. Further research is needed to discover more contributing factors in the pathological alterations of alcohol addiction to offer sex-specific biomarkers and treatment.

The Study of the Association of Polymorphisms in LSP1, GPNMB, PDPN, TAGLN, TSPO, and TUBB6 Genes with the Risk and Outcome of Ischemic Stroke in the Russian Population.

To date, there has been great progress in understanding the genetic basis of ischemic stroke (IS); however, several aspects of the condition remain underexplored, including the influence of genetic factors on post-stroke outcomes and the identification of causative loci. We proposed that an analysis of the results obtained from animal models of brain ischemia could be helpful. To this end, we developed a bioinformatic approach for exploring single-nucleotide polymorphisms (SNPs) in human orthologs of rat genes expressed differentially after induced brain ischemia. Using this approach, we identified and analyzed 11 SNPs from 6 genes in 553 Russian individuals (331 patients with IS and 222 controls). We assessed the association of SNPs with the risk of IS and IS outcomes. We found that the SNPs rs858239 (GPNMB), rs907611 (LSP1), and rs494356 (TAGLN) were associated with different parameters of IS functional outcomes. In addition, the SNP rs1261025 (PDPN) was associated significantly with IS itself (p = 0.0188, recessive model). All these associations were demonstrated for the first time. Analysis of the literature suggests that they should be characterized as being inflammation related. This supports the pivotal role of inflammation in both the incidence of stroke and post-stroke outcomes. We believe the findings reported here will help with stroke prognosis in the future.

Gamma-Aminobutyric Acid Type A Receptor Variants are Associated with Autism Spectrum Disorders.

Despite several efforts to identify the causes of autism spectrum disorders (ASD), its etiology remains still unclear. Among other aspects, genes that encode neurotransmitter receptors are strong candidates for autism. Here, we wanted to study some genetic variants of gamma-aminobutyric acid (GABA) receptor subunit genes GABRB3, GABRG3, and GABRA5, located on chromosome 15q11-q13 that might contribute to the etiology of ASD in the affected children of West Bengal. rs7180158, rs2081648 (GABRB3); rs12910555 (GABRG3); rs35399885, rs35832850 (GABRA5) were analyzed in 316 children with ASD and 227 healthy controls. Phenotypic associations were evaluated by Childhood Autism Rating Scale (CARS). Gene expression levels were measured by quantitative real-time PCR. ASD probands showed a higher frequency of "A" allele for rs7180158, "G" allele for rs12901555, and "T" allele for rs35399885. The GA + AA genotypes (rs7180158) and CT + TT genotypes (rs35399885) were found to confer significant risk towards ASD. rs2081648 was found to have transmission bias in the family. Additionally, these variants were found to be associated with one or more of ASD-associated phenotypic traits. Multifactor dimensionality reduction (MDR) analyses showed mostly independent contributory effects of some of the variants. Again, the gene expression levels of GABRB3, GABRG3, and GABRA5 were downregulated in the cases than the controls. ForGABRA5 rs35399885, the CC genotypes corresponded to higher expression levels compared to the other groups. This study reveals that genetic variants of GABAA receptor subunit genes are significantly associated with ASD. No data for the mentioned variants are found in the population of West Bengal, India.

Impact of Translocator Protein 18 kDa (TSPO) Deficiency on Mitochondrial Function and the Inflammatory State of Human C20 Microglia Cells.

Microglia are the resident immune cells of the central nervous system. Upon stimulus presentation, microglia polarize from a resting to an activated state. Microglial translocator protein 18 kDa (TSPO) is considered a marker of inflammation. Here, we characterized the role of TSPO by investigating the impact of TSPO deficiency on human microglia. We used TSPO knockout (TSPO-/-) variants of the human C20 microglia cell line. We found a significant reduction in the TSPO-associated protein VDAC1 in TSPO-/- cells compared to control cells. Moreover, we assessed the impact of TSPO deficiency on calcium levels and the mitochondrial membrane potential. Cytosolic and mitochondrial calcium concentrations were increased in TSPO-/- cell lines, whereas the mitochondrial membrane potential tended to be lower. Assessment of the mitochondrial DNA copy number via RT-PCR revealed a decreased amount of mtDNA in the TSPO-/- cells when compared to controls. Moreover, the metabolic profiles of C20 cells were strongly dependent on the glycolytic pathway. However, TSPO depletion did not affect the cellular metabolic profile. Measurement of the mRNA expression levels of the pro-inflammatory mediators revealed an attenuated response to pro-inflammatory stimuli in TSPO-depleted cells, implying a role for the TSPO protein in the process of microglial polarization.

Association of GABA receptor delta subunit gene variations with increased risk of methamphetamine dependence.

OBJECTIVE: Evidence reveals that γ-aminobutyric acid (GABA) receptors are involved in the development of methamphetamine (METH) dependence. The GABA receptor delta subunit gene (GABRD) might be a good candidate gene for METH dependence. In a case-control study, we investigated the association between the single nucleotide polymorphisms (SNPs) in GABRD and METH dependence in a Chinese Han population. METHODS: A total of 300 METH dependent patients and 300 age and sex matched normal control subjects were recruited. Four SNPs (rs13303344, rs4481796, rs2376805, and rs2229110) in GABRD were determined with the TaqMan genotyping assay. The association of the SNPs with METH dependence was assessed. RESULTS: Only the allele frequency of rs2376805 significantly differed between the patients and controls (P = 0.030). The G allele frequency of rs2376805 was higher in the METH dependent group than in the controls (odds ratio = 1.332, 95 % CI: 1.028-1.724). This association was found in females but not in males. In females, the frequencies of genotype and allele at rs2376805 significantly differed between the patients and controls (P = 0.025, 0.022, respectively); the rs2376805 G allele may also be a risk factor for METH dependence (odds ratio = 1.548, 95 % CI: 1.063-2.257). The haplotype ACGT frequency significantly differed between the patients and controls in total subjects (P = 0.008, odds ratio = 1.815, 95 % CI: 1.183-2.782), as well as in females (P = 0.005, odds ratio = 2.702, 95 % CI: 1.313-5.562). In females only, the METH craving score was significantly lower in patients harboring the G allele at rs2376805 than in those harboring the homozygous AA genotype (P = 0.044). CONCLUSION: The preliminary results indicate that GABRD rs2376805 is associated with METH dependence, especially in females.

Liver saturated fat content associates with hepatic DNA methylation in obese individuals.

BACKGROUND: Accumulation of saturated fatty acids (SFAs) in the liver is known to induce hepatic steatosis and inflammation causing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although SFAs have been shown to affect the epigenome in whole blood, pancreatic islets, and adipose tissue in humans, and genome-wide DNA methylation studies have linked epigenetic changes to NAFLD and NASH, studies focusing on the association of SFAs and DNA methylation in human liver are missing. We, therefore, investigated whether human liver SFA content associates with DNA methylation and tested if SFA-linked alterations in DNA methylation associate with NAFLD-related clinical phenotypes in obese individuals. RESULTS: We identified DNA methylation (Infinium HumanMethylation450 BeadChip) of 3169 CpGs to be associated with liver total SFA content (q-value < 0.05) measured using proton NMR spectroscopy in participants of the Kuopio Obesity Surgery Study (n = 51; mean ± SD:49.3 ± 8.5 years old; BMI:43.7 ± 6.2 kg/m2). Of these 3169 sites, 797 overlapped with previously published NASH-associated CpGs (NASH-SFA), while 2372 CpGs were exclusively associated with SFA (Only-SFA). The corresponding annotated genes of these only-SFA CpGs were found to be enriched in pathways linked to satiety and hunger. Among the 54 genes mapping to these enriched pathways, DNA methylation of CpGs mapping to PRKCA and TSPO correlated with their own mRNA expression (HumanHT-12 Expression BeadChip). In addition, DNA methylation of another ten of these CpGs correlated with the mRNA expression of their neighboring genes (p value < 0.05). The proportion of CpGs demonstrating a correlation of DNA methylation with plasma glucose was higher in NASH-SFA and only-SFA groups, while the proportion of significant correlations with plasma insulin was higher in only-NASH and NASH-SFA groups as compared to all CpGs on the Illumina 450 K array (Illumina, San Diego, CA, USA). CONCLUSIONS: Our results suggest that one of the mechanisms how SFA could contribute to metabolic dysregulation in NAFLD is at the level of DNA methylation. We further propose that liver SFA-related DNA methylation profile may contribute more to hyperglycemia, while insulin-related methylation profile is more linked to NAFLD or NASH. Further research is needed to elucidate the molecular mechanisms behind these observations.

[Clinical Value of Translocator Protein Gene in Evaluating the Efficacy of FLT3-ITD/DNMT3A R882 Double-Mutated Acute Myeloid Leukemia].

OBJECTIVE: To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML). METHODS: Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation). RESULTS: The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011). CONCLUSION: TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.

Radiation-induced neuroinflammation monitoring by the level of peripheral blood monocytes with high expression of translocator protein.

PURPOSE: Currently there are no effective diagnostic methods for the control of neuroinflammation before manifestation of cognitive impairment after head irradiation. The translocator protein (TSPO) is highly expressed in glial cells upon brain damage, therefore we compared the changes in the number of cells with high TSPO expression in the brain and peripheral blood during radiation-induced neuroinflammation. MATERIALS AND METHODS: Hippocampal cytokines mRNA expression and the content of cells with high TSPO expression in the brain and peripheral blood monocytes were analyzed up to eight months after mice head γ-irradiation at a dose of 2 Gy or 8Gy. RESULTS: Mice irradiation at a dose of 8 Gy causes neuroinflammation, accompanied by an increase of M1 microglia and TSPOhigh cells in the brain, elevated gene expression of pro-inflammatory and decreased of anti-inflammatory cytokines along with an increased number of microglia and astrocytes in the hippocampus. The content of TSPOhigh cells in the brain correlates with the level TSPOhigh monocytes in three days, one month and two months after exposure. CONCLUSIONS: An increase in the level of the monocytes with high expression of TSPO may be considered as a marker for an early diagnostics of post-radiation brain damage leading to cognitive impairment.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.