Hydroxymethylglutaryl-CoA reductase activity is essential for mitochondrial ß-oxidation of fatty acids to prevent lethal accumulation of long-chain acylcarnitines in the mouse liver.

BACKGROUND AND PURPOSE: Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), and exert adverse effects on mitochondrial function, although the mechanisms underlying these effects remain unclear. We used a tamoxifen-induced Hmgcr-knockout (KO) mouse model, a multi-omics approach and mitochondrial function assessments to investigate whether decreased HMGCR activity impacts key liver energy metabolism pathways. EXPERIMENTAL APPROACH: We established a new mouse strain using the Cre/loxP system, which enabled whole-body deletion of Hmgcr expression. These mice were crossed with Rosa26Cre mice and treated with tamoxifen to delete Hmgcr in all cells. We performed transcriptomic and metabolomic analyses and thus evaluated time-dependent changes in metabolic functions to identify the pathways leading to cell death in Hmgcr-KO mice. KEY RESULTS: Lack of Hmgcr expression resulted in lethality, due to acute liver damage caused by rapid disruption of mitochondrial fatty acid ß-oxidation and very high accumulation of long-chain (LC) acylcarnitines in both male and female mice. Gene expression and KO-related phenotype changes were not observed in other tissues. The progression to liver failure was driven by diminished peroxisome formation, which resulted in impaired mitochondrial and peroxisomal fatty acid metabolism, enhanced glucose utilization and whole-body hypoglycaemia. CONCLUSION AND IMPLICATIONS: Our findings suggest that HMGCR is crucial for maintaining energy metabolism balance, and its activity is necessary for functional mitochondrial ß-oxidation. Moreover, statin-induced adverse reactions might be rescued by the prevention of LC acylcarnitine accumulation.

Fluvastatin-induced myofibrillar damage is associated with elevated ROS, and impaired fatty acid oxidation, and is preceded by mitochondrial morphological changes.

Previously, we showed that fluvastatin treatment induces myofibrillar damage and mitochondrial phenotypes in the skeletal muscles of Drosophila. However, the sequential occurrence of mitochondrial phenotypes and myofibril damage remains elusive. To address this, we treated flies with fluvastatin for two and five days and examined their thorax flight muscles using confocal microscopy. In the two-day fluvastatin group, compared to the control, thorax flight muscles exhibited mitochondrial morphological changes, including fragmentation, rounding up and reduced content, while myofibrils remained organized in parallel. In the five-day fluvastatin treatment, not only did mitochondrial morphological changes become more pronounced, but myofibrils became severely disorganized with significantly increased thickness and spacing, along with myofilament abnormalities, suggesting myofibril damage. These findings suggest that fluvastatin-induced mitochondrial changes precede myofibril damage. Moreover, in the five-day fluvastatin group, the mitochondria demonstrated elevated H2O2 and impaired fatty acid oxidation compared to the control group, indicating potential mitochondrial dysfunction. Surprisingly, knocking down Hmgcr (Drosophila homolog of HMGCR) showed normal mitochondrial respiration in all parameters compared to controls or five-day fluvastatin treatment, which suggests that fluvastatin-induced mitochondrial dysfunction might be independent of Hmgcr inhibition. These results provide insights into the sequential occurrence of mitochondria and myofibril damage in statin-induced myopathy for future studies.

Drosophila as a Rapid Screening Model to Evaluate the Hypoglycemic Effects of Dipeptidyl Peptidase 4 (DPP4) Inhibitors: High Evolutionary Conservation of DPP4.

Dipeptidyl peptidase 4 (DPP4) inhibitors, commonly known as gliptins, have been an integral part of the treatment of type 2 diabetes mellitus (T2DM) for several years. Despite their remarkable efficacy in lowering glucose levels and their compatibility with other hypoglycemic drugs, recent studies have revealed adverse effects, prompting the search for improved drugs within this category, which has required the use of animal models to verify the hypoglycemic effects of these compounds. Currently, in many countries the use of mammals is being significantly restricted, as well as cost prohibitive, and alternative in vivo approaches have been encouraged. In this sense, Drosophila has emerged as a promising alternative for several compelling reasons: it is cost-effective, offers high experimental throughput, is genetically manipulable, and allows the assessment of multigenerational effects, among other advantages. In this study, we present evidence that diprotin A, a DPP4 inhibitor, effectively reduces glucose levels in Drosophila hemolymph. This discovery underscores the potential of Drosophila as an initial screening tool for novel compounds directed against DPP4 enzymatic activity.

Vesicular glutamate transporter 2 expression in the ventral tegmental area of outbred male rats following exposure to nicotine and alcohol.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Differential Blood-Brain Barrier Transport and Cell Uptake of Cyclic Peptides In Vivo and In Vitro.

The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, Kp,uu,brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.

SLC38A10 Deficiency in Mice Affects Plasma Levels of Threonine and Histidine in Males but Not in Females: A Preliminary Characterization Study of SLC38A10-/- Mice.

Solute carriers belong to the biggest group of transporters in the human genome, but more knowledge is needed to fully understand their function and possible role as therapeutic targets. SLC38A10, a poorly characterized solute carrier, is preliminary characterized here. By using a knockout mouse model, we studied the biological effects of SLC38A10 deficiency in vivo. We performed a transcriptomic analysis of the whole brain and found seven differentially expressed genes in SLC38A10-deficient mice (Gm48159, Nr4a1, Tuba1c, Lrrc56, mt-Tp, Hbb-bt and Snord116/9). By measuring amino acids in plasma, we found lower levels of threonine and histidine in knockout males, whereas no amino acid levels were affected in females, suggesting that SLC38A10-/- might affect sexes differently. Using RT-qPCR, we investigated the effect of SLC38A10 deficiency on mRNA expression of other SLC38 members, Mtor and Rps6kb1 in the brain, liver, lung, muscle, and kidney, but no differences were found. Relative telomere length measurement was also taken, as a marker for cellular age, but no differences were found between the genotypes. We conclude that SLC38A10 might be important for keeping amino acid homeostasis in plasma, at least in males, but no major effects were seen on transcriptomic expression or telomere length in the whole brain.

Statins Induce Locomotion and Muscular Phenotypes in Drosophila melanogaster That Are Reminiscent of Human Myopathy: Evidence for the Role of the Chloride Channel Inhibition in the Muscular Phenotypes.

The underlying mechanisms for statin-induced myopathy (SIM) are still equivocal. In this study, we employ Drosophila melanogaster to dissect possible underlying mechanisms for SIM. We observe that chronic fluvastatin treatment causes reduced general locomotion activity and climbing ability. In addition, transmission microscopy of dissected skeletal muscles of fluvastatin-treated flies reveals strong myofibrillar damage, including increased sarcomere lengths and Z-line streaming, which are reminiscent of myopathy, along with fragmented mitochondria of larger sizes, most of which are round-like shapes. Furthermore, chronic fluvastatin treatment is associated with impaired lipid metabolism and insulin signalling. Mechanistically, knockdown of the statin-target Hmgcr in the skeletal muscles recapitulates fluvastatin-induced mitochondrial phenotypes and lowered general locomotion activity; however, it was not sufficient to alter sarcomere length or elicit myofibrillar damage compared to controls or fluvastatin treatment. Moreover, we found that fluvastatin treatment was associated with reduced expression of the skeletal muscle chloride channel, ClC-a (Drosophila homolog of CLCN1), while selective knockdown of skeletal muscle ClC-a also recapitulated fluvastatin-induced myofibril damage and increased sarcomere lengths. Surprisingly, exercising fluvastatin-treated flies restored ClC-a expression and normalized sarcomere lengths, suggesting that fluvastatin-induced myofibrillar phenotypes could be linked to lowered ClC-a expression. Taken together, these results may indicate the potential role of ClC-a inhibition in statin-associated muscular phenotypes. This study underlines the importance of Drosophila melanogaster as a powerful model system for elucidating the locomotion and muscular phenotypes, promoting a better understanding of the molecular mechanisms underlying SIM.

SLC38A10 Knockout Mice Display a Decreased Body Weight and an Increased Risk-Taking Behavior in the Open Field Test.

The solute carrier 38 family (SLC38) is a family of 11 members. The most common substrate among these are alanine and glutamine, and members are present in a wide range of tissues with important functions for several biological processes, such as liver and brain function. Some of these transporters are better characterized than others and, in this paper, a behavioral characterization of SLC38A10-/- mice was carried out. A battery of tests for general activity, emotionality, motor function, and spatial memory was used. Among these tests, the elevated plus maze, Y-maze, marble burying and challenging beam walk have not been tested on the SLC38A10-/- mice previously, while the open field and the rotarod tests have been performed by the International Mouse Phenotyping Consortium (IMPC). Unlike the results from IMPC, the results from this study showed that SLC38A10-/- mice spend less time in the wall zone in the open field test than WT mice, implying that SLC38A10-deficient mice have an increased explorative behavior, which suggests an important function of SLC38A10 in brain. The present study also confirmed IMPC's data regarding rotarod performance and weight, showing that SLC38A10-/- mice do not have an affected motor coordination impairment and have a lower body weight than both SLC38A10+/- and SLC38A10+/+ mice. These results imply that a complete deficiency of the SLC38A10 protein might affect body weight homeostasis, but the underlying mechanisms needs to be studied further.

SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.

Glutamate acts as a critical regulator of neurotransmitter balance, recycling, synaptic function and homeostasis in the brain and glutamate transporters control glutamate levels in the brain. SLC38A10 is a member of the SLC38 family and regulates protein synthesis and cellular stress responses. Here, we uncover the role of SLC38A10 as a transceptor involved in glutamate-sensing signaling pathways that control both the glutamate homeostasis and mTOR-signaling. The culture of primary cortex cells from SLC38A10 knockout mice had increased intracellular glutamate. In addition, under nutrient starvation, KO cells had an impaired response in amino acid-dependent mTORC1 signaling. Combined studies from transcriptomics, protein arrays and metabolomics established that SLC38A10 is involved in mTOR signaling and that SLC38A10 deficient primary cortex cells have increased protein synthesis. Metabolomic data showed decreased cholesterol levels, changed fatty acid synthesis, and altered levels of fumaric acid, citrate, 2-oxoglutarate and succinate in the TCA cycle. These data suggests that SLC38A10 may act as a modulator of glutamate homeostasis, and mTOR-sensing and loss of this transceptor result in lower cholesterol, which could have implications in neurodegenerative diseases.

Paracetamol (Acetaminophen) and its Effect on the Developing Mouse Brain.

Paracetamol, or acetaminophen (AAP), is the most commonly used analgesic during pregnancy and early life. While therapeutic doses of AAP are considered harmless during these periods, recent findings in both humans and rodents suggest a link between developmental exposure to AAP and behavioral consequences later in life. The aim of this study is to evaluate the impact of neonatal exposure to clinically relevant doses of AAP on adult spontaneous behavior, habituation, memory, learning, and cognitive flexibility later in life using a mouse model. Markers of oxidative stress, axon outgrowth, and glutamatergic transmission were also investigated in the hippocampus during the first 24 h after exposure. In addition, potential long-term effects on synaptic density in the hippocampus have been investigated. In a home cage setting, mice neonatally exposed to AAP (30 + 30 mg/kg, 4 h apart) on postnatal day 10 displayed altered spontaneous behavior and changed habituation patterns later in life compared to controls. These mice also displayed reduced memory, learning and cognitive flexibility compared to control animals in the Morris water maze. An increase of markers for oxidative stress was observed in the hippocampus 6 h after AAP exposure. As AAP is the first choice treatment for pain and/or fever during pregnancy and early life, these results may be of great importance for risk assessment. Here we show that AAP can have persistent negative effects on brain development and suggest that AAP, despite the relatively low doses, is capable to induce acute oxidative stress in the hippocampus.

Behavioral profiling of SLC38A10 knockout mice using the multivariate concentric square fieldTM test.

Introduction: SLC38A10 is a gene that encodes the SLC38A10 protein, also known as SNAT10. The SLC38 family is evolutionary old, and SLC38A10 is one of the oldest members of the family. It is ubiquitously expressed, and its substrates are glutamine, glutamate, alanine, aspartate, and serine. However, little is known about its biological importance. Methods: In the current study, an SLC38A10 knockout mouse was run in the multivariate concentric square field TM (MCSF) test. The MCSF test gives the mouse a choice of areas to explore; sheltered areas, elevated and illuminated areas, or open spaces, and a behavioral profile is obtained. The multivariate data obtained were analyzed (i) for each parameter, (ii) parameters grouped into functional categories, and (iii) with a principal component analysis. Results: In the trend analysis, knockout mice had a decreased exploratory behavior compared to controls but did not show a distinct grouping in the principal component analysis. Discussion: There was not a pronounced difference in the behavioral profile in SLC38A10 knockout mice compared to their wild-type controls, although subtle alterations in zones associated with exploratory behavior and risk assessment in female and male knockout mice, respectively, could be observed. These results imply that a loss of function of the SLC38A10 protein in mice does not drastically alter behavior in the MSCF test.

A phylogenetic analysis between humans and D. melanogaster: A repertoire of solute carriers in humans and flies.

The solute carrier (SLC) superfamily is the largest group of transporters in humans, with the role to transport solutes across plasma membranes. The SLCs are currently divided into 65 families with 430 members. Here, we performed a detailed mining of the SLC superfamily and the recent annotated family of "atypical" SLCs in human and D. melanogaster using Hidden Markov Models and PSI-BLAST. Our analyses identified 381 protein sequences in D. melanogaster and of those, 55 proteins have not been previously identified in flies. In total, 11 of the 65 human SLC families were found to not be conserved in flies, while a few families are highly conserved, which perhaps reflects the families' functions and roles in cellular pathways. This study provides the first collection of all SLC sequences in D. melanogaster and can serve as a SLC database to be used for classification of SLCs in other phyla.

The Fly Homologue of MFSD11 Is Possibly Linked to Nutrient Homeostasis and Has a Potential Role in Locomotion: A First Characterization of the Atypical Solute Carrier CG18549 in Drosophila Melanogaster.

Cellular transport and function are dependent on substrate influx and efflux of various compounds. In humans, the largest superfamily of transporters is the SoLute Carriers (SLCs). Many transporters are orphans and little to nothing is known about their expression and/or function, yet they have been assigned to a cluster called atypical SLCs. One of these atypical SLCs is MFSD11. Here we present a first in-depth characterization of the MFSD11, CG18549. By gene expression and behavior analysis on ubiquitous and brain-specific knockdown flies. CG18549 knockdown flies were found to have altered adipokinetic hormone and adipokinteic hormone receptor expression as well as reduced vesicular monoamine transporter expression; to exhibit an altered locomotor behavior, and to have an altered reaction to stress stimuli. Furthermore, the gene expression of CG18549 in the brain was visualized and abundant expression in both the larvae and adult brain was observed, a result that is coherent with the FlyAtlas Anatomy microarray. The exact mechanism behind the observed behaviors is not fully understood, but this study provides new insights into the expression and function of CG18549. Clearly, these results provide a strong example as to why it is vital to fully characterize orphan transporters and through that gain knowledge about the body during normal condition and disease.

Integrating Statistical and Machine-Learning Approach for Meta-Analysis of Bisphenol A-Exposure Datasets Reveals Effects on Mouse Gene Expression within Pathways of Apoptosis and Cell Survival.

Bisphenols are important environmental pollutants that are extensively studied due to different detrimental effects, while the molecular mechanisms behind these effects are less well understood. Like other environmental pollutants, bisphenols are being tested in various experimental models, creating large expression datasets found in open access storage. The meta-analysis of such datasets is, however, very complicated for various reasons. Here, we developed an integrating statistical and machine-learning model approach for the meta-analysis of bisphenol A (BPA) exposure datasets from different mouse tissues. We constructed three joint datasets following three different strategies for dataset integration: in particular, using all common genes from the datasets, uncorrelated, and not co-expressed genes, respectively. By applying machine learning methods to these datasets, we identified genes whose expression was significantly affected in all of the BPA microanalysis data tested; those involved in the regulation of cell survival include: Tnfr2, Hgf-Met, Agtr1a, Bdkrb2; signaling through Mapk8 (Jnk1)); DNA repair (Hgf-Met, Mgmt); apoptosis (Tmbim6, Bcl2, Apaf1); and cellular junctions (F11r, Cldnd1, Ctnd1 and Yes1). Our results highlight the benefit of combining existing datasets for the integrated analysis of a specific topic when individual datasets are limited in size.

Toll-like receptor 4 methylation grade is linked to depressive symptom severity.

This study explores potential associations between the methylation of promoter-associated CpG sites of the toll-like receptor (TLR)-family, plasma levels of pro-inflammatory proteins and depressive symptoms in young female psychiatric patients. Ratings of depressive symptoms and blood samples were obtained from 92 young women seeking psychiatric care. Methylation of 32 promoter-associated CpG sites in TLR1 to TLR10 was analysed using the Illumina Infinium Methylation EPIC BeadChip. Expression levels of 91 inflammatory proteins were determined by proximity extension assay. Statistical correlations between depressive state, TLR1-10 methylation and inflammatory proteins were investigated. Four additional cohorts were studied to evaluate the generalizability of the findings. In the discovery cohort, methylation grade of cg05429895 (TLR4) in blood was inversely correlated with depressive symptoms score in young adults. After correction for multiple testing, plasma levels of macrophage inflammatory protein 1ß (MIP-1ß/CCL4) were associated with both TLR4 methylation and depressive symptom severity. A similar inverse association between TLR4 methylation in blood and affective symptoms score was also found in a cohort of 148 both males and females (<40 years of age) from the Danish Twin Registry. These findings were not, however, replicated in three other external cohorts; which differed from the first two cohorts by a higher age and mixed ethnicities, thus limiting the generalizability of our findings. However, TLR4 methylation inversely correlated with TLR4 mRNA expression in the Danish Twin Study indicating a functional significance of methylation at this particular CpG. Higher depression scores in young Scandinavian adults was associated with decreased methylation of TLR4 in blood.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.

SLC38A10 Transporter Plays a Role in Cell Survival Under Oxidative Stress and Glutamate Toxicity.

Solute carrier (SLC) transporters regulate amino acids, glucose, ions, and metabolites that flow across cell membranes. In the brain, SLCs are the key regulators of neurotransmission, in particular, the glutamate/GABA-glutamine (GGG) cycle. Genetic mutations in SLCs are associated with various neurodevelopmental and neurodegenerative diseases. In this study, we have investigated the role of SLC38A10 under acute oxidative and glutamate stress in mouse primary cortical cells from SLC38A10 knockout (KO) mice. The ER/golgi localized transporter, SLC38A10, transports glutamate, glutamine, and alanine in brain cells, and the aim of this study was to determine the possible effects of removal of SLC38A10 in primary cortical cells under glutamate and oxidative challenges. Primary cortical neuronal cultures of wild-type (WT) cell and SLC38A10 KO mice were subjected to different concentrations of glutamate and hydrogen peroxide. There was no morphological change observed between KO and WT cortical neurons in culture. Interestingly, KO cells showed significantly lower cell viability and higher cell death compared to WT cells under both glutamate and hydrogen peroxide exposure. Further, we evaluated the possible role of p53 in neuronal cell apoptosis in KO cells. We found decreased intracellular p53 protein levels under glutamate and hydrogen peroxide treatment in KO cortical cells. In contrast, caspase 3/7 activity remains unaltered under all conditions. These results demonstrate an indirect relationship between the expression of SLC38A10 and p53 and a role in the cell defense mechanism against neurotoxicity.

Differentiation of two human neuroblastoma cell lines alters SV2 expression patterns.

BACKGROUND: The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation. METHODS: Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed. RESULTS: The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells. CONCLUSION: The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function.

Glutamine Uptake via SNAT6 and Caveolin Regulates Glutamine-Glutamate Cycle.

SLC38A6 (SNAT6) is the only known member of the SLC38 family that is expressed exclusively in the excitatory neurons of the brain. It has been described as an orphan transporter with an unknown substrate profile, therefore very little is known about SNAT6. In this study, we addressed the substrate specificity, mechanisms for internalization of SNAT6, and the regulatory role of SNAT6 with specific insights into the glutamate-glutamine cycle. We used tritium-labeled amino acids in order to demonstrate that SNAT6 is functioning as a glutamine and glutamate transporter. SNAT6 revealed seven predicted transmembrane segments in a homology model and was localized to caveolin rich sites at the plasma membrane. SNAT6 has high degree of specificity for glutamine and glutamate. Presence of these substrates enables formation of SNAT6-caveolin complexes that aids in sodium dependent trafficking of SNAT6 off the plasma membrane. To further understand its mode of action, several potential interacting partners of SNAT6 were identified using bioinformatics. Among them where CTP synthase 2 (CTPs2), phosphate activated glutaminase (Pag), and glutamate metabotropic receptor 2 (Grm2). Co-expression analysis, immunolabeling with co-localization analysis and proximity ligation assays of these three proteins with SNAT6 were performed to investigate possible interactions. SNAT6 can cycle between cytoplasm and plasma membrane depending on availability of substrates and interact with Pag, synaptophysin, CTPs2, and Grm2. Our data suggest a potential role of SNAT6 in glutamine uptake at the pre-synaptic terminal of excitatory neurons. We propose here a mechanistic model of SNAT6 trafficking that once internalized influences the glutamate-glutamine cycle in presence of its potential interacting partners.

Evaluation of the dentate gyrus in adult mice exposed to acetaminophen (paracetamol) on postnatal day 10.

Acetaminophen (AAP; or paracetamol) is a widely used nonprescription drug with antipyretic and analgesic properties. Alarmingly, there is an increasing body of evidence showing that developmental exposure to AAP is associated with adverse behavioural outcomes later in life. We have previously shown that relevant doses of AAP in 10-day-old mice affected memory, learning and locomotor activity in the adult animals. Interestingly, the neurons of the dentate gyrus (DG) have a relatively late time of origin as they are generated during the first two weeks of postnatal life in rodents. Since the generation of these cells, which are important for memory processing, coincides with our AAP exposure, we aim to investigate if the cytoarchitecture of the DG is affected by postnatal day 10 AAP treatment. In addition, we investigate if markers for differentiation and migration in the hippocampus were affected by the same treatment. We did not observe any visual effects in adult DG cytoarchitecture, nor any changes of markers for differentiation/migration in the hippocampus in 24 hr after exposure. Even though a large effect size was estimated on adult DG thickness following AAP exposure, the estimated 95% CIs around the differences of the means reveal no significant effect. Hence, larger sample sizes are warranted to clarify if neonatal AAP exposure affects adult DG thickness in mice.