ABSTRACT
Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1ß treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.
Subject(s)
Escherichia coli Infections , Intestinal Mucosa , Animals , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Mice , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Caco-2 Cells , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Escherichia coli , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/pathology , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/genetics , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Creatine/metabolismABSTRACT
X-linked creatine transporter deficiency is caused by hemizygous or heterozygous pathogenic variants in SLC6A8 that cause neuropsychiatric symptoms because of impaired uptake of creatine into tissues throughout the body. Small cohorts have suggested that supplementation of creatine, arginine, and glycine can stop disease progression in males, but only six cases of supplementation in females have been published. Here, we present a female with a de-novo pathogenic SLC6A8 variant who had ongoing weight loss, mild intellectual disability, and neuropsychiatric symptoms. Magnetic resonance spectroscopy of the brain showed reduced creatine on all acquired spectra. The patient was started on creatine-monohydrate, l -arginine, and l -glycine supplementation, and she had significant symptomatic improvement within the following 3 weeks. After 8 months of supplementation, magnetic resonance spectroscopy showed improved creatine concentrations with normalizing semiquantitative ratios with other brain metabolites. Current data supports clinicians trialing creatine, arginine, and glycine supplements for female patients with creatine transporter deficiency.
Subject(s)
Arginine , Creatine , Dietary Supplements , Glycine , Mental Retardation, X-Linked , Plasma Membrane Neurotransmitter Transport Proteins , Humans , Female , Creatine/metabolism , Creatine/deficiency , Glycine/metabolism , Arginine/metabolism , Arginine/therapeutic use , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/drug therapy , Mental Retardation, X-Linked/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Brain/metabolism , Adult , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Magnetic Resonance Spectroscopy , Intellectual Disability/genetics , Intellectual Disability/metabolism , Intellectual Disability/drug therapy , Brain Diseases, Metabolic, Inborn , Membrane Transport ProteinsABSTRACT
INTRODUCTION: Lung cancer is a major cause of cancer-related death worldwide and effective therapies, besides surgery, are available only for a small proportion of patients. Since cellular respiration is known to be broadly altered in malignant tumors, the cellular processes of respiration can be a potential therapeutic target. One important element of cellular respiration is creatine and its transport by the creatine transporter SLC6A8. Here we describe the expression of SLC6A8 at the RNA and protein level, epigenetic modifications as well as survival analysis in NSCLC tissues and matched controls. MATERIALS AND METHODS: We analyzed epigenetic modifications of the SLC68A gene in 32 patients, of which 18 were additionally analyzed by transcriptome analysis. The expression of SLC6A8 at the protein level was assessed by immunohistochemistry using an independent cohort and correlated with clinicopathological data including survival. Kaplan-Meier analysis was performed to analyze the possible effects of the transcriptional levels of SLC6A8 in another separate cohort (n=1925). RESULTS: SLC6A8 loci are epigenetically modified in NSCLC compared with tumor-free controls. SLC6A8 is upregulated in NSCLC at the RNA and protein level. High mRNA expression of SLC6A8 was associated with an overall poor prognosis in lung adenocarcinoma patients and displayed the strongest adverse prognostic effect in male smokers with adenocarcinomas. Results of transcriptome analysis were partially confirmed at the protein level. CONCLUSIONS: Our results suggest an important role of creatine and its transport via SLC6A8 in NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Epigenesis, Genetic , Lung Neoplasms , Up-Regulation , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Male , Female , Middle Aged , Aged , Gene Expression Regulation, Neoplastic , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Prognosis , Kaplan-Meier Estimate , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Adult , Membrane Transport ProteinsABSTRACT
The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.
Subject(s)
Sodium , Symporters , Sodium/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Symporters/metabolism , Binding Sites , Neurotransmitter AgentsABSTRACT
Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.
Subject(s)
Creatine , Mutation, Missense , Humans , Creatine/metabolism , Mutation , Biological Transport , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolismABSTRACT
The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 - 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.
Subject(s)
Molecular Conformation , Neurotransmitter Agents , Symporters/chemistry , Temperature , Kinetics , Membrane Proteins , Molecular Dynamics Simulation , Pharmaceutical Preparations , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Protein Conformation , SodiumABSTRACT
Acute myeloid leukaemia (AML) carrying nucleophosmin (NPM1) mutations has been defined as a distinct entity of acute leukaemia. Despite remarkable improvements in diagnosis and treatment, the long-term outcomes for this entity remain unsatisfactory. Emerging evidence suggests that leukaemia, similar to other malignant diseases, employs various mechanisms to evade killing by immune cells. However, the mechanism of immune escape in NPM1-mutated AML remains unknown. In this study, both serum and leukemic cells from patients with NPM1-mutated AML impaired the immune function of CD8+ T cells in a co-culture system. Mechanistically, leukemic cells secreted miR-19a-3p into the tumour microenvironment (TME) via small extracellular vesicles (sEVs), which was controlled by the NPM1-mutated protein/CCCTC-binding factor (CTCF)/poly (A)-binding protein cytoplasmic 1 (PABPC1) signalling axis. sEV-related miR-19a-3p was internalized by CD8+ T cells and directly repressed the expression of solute-carrier family 6 member 8 (SLC6A8; a creatine-specific transporter) to inhibit creatine import. Decreased creatine levels can reduce ATP production and impair CD8+ T cell immune function, leading to immune escape by leukemic cells. In summary, leukemic cell-derived sEV-related miR-19a-3p confers immunosuppression to CD8+ T cells by targeting SLC6A8-mediated creatine import, indicating that sEV-related miR-19a-3p might be a promising therapeutic target for NPM1-mutated AML.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Creatine/metabolism , Extracellular Vesicles/metabolism , Immune Tolerance , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Mutation , Nerve Tissue Proteins/metabolism , Nucleophosmin/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Signal Transduction/immunology , Adult , Aged , Biological Transport , Coculture Techniques/methods , Female , Humans , Leukemia, Myeloid, Acute/blood , Male , MicroRNAs/metabolism , Middle Aged , Tumor Escape , Tumor Microenvironment/immunologyABSTRACT
Creatine (Cr) Transporter Deficiency (CTD) is an X-linked metabolic disorder, mostly caused by missense mutations in the SLC6A8 gene and presenting with intellectual disability, autistic behavior, and epilepsy. There is no effective treatment for CTD and patients need lifelong assistance. Thus, the research of novel intervention strategies is a major scientific challenge. Animal models are an excellent tool to dissect the disease pathogenetic mechanisms and drive the preclinical development of therapeutics. This review illustrates the current knowledge about Cr metabolism and CTD clinical aspects, with a focus on mainstay diagnostic and therapeutic options. Then, we discuss the rodent models of CTD characterized in the last decade, comparing the phenotypes expressed within clinically relevant domains and the timeline of symptom development. This analysis highlights that animals with the ubiquitous deletion/mutation of SLC6A8 genes well recapitulate the early onset and the complex pathological phenotype of the human condition. Thus, they should represent the preferred model for preclinical efficacy studies. On the other hand, brain- and cell-specific conditional mutants are ideal for understanding the basis of CTD at a cellular and molecular level. Finally, we explain how CTD models might provide novel insight about the pathogenesis of other disorders, including cancer.
Subject(s)
Brain Diseases, Metabolic, Inborn/pathology , Brain Diseases, Metabolic, Inborn/therapy , Central Nervous System/pathology , Creatine/deficiency , Disease Models, Animal , Mental Retardation, X-Linked/pathology , Mental Retardation, X-Linked/therapy , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Animals , Biomarkers/metabolism , Brain Diseases, Metabolic, Inborn/metabolism , Creatine/metabolism , Humans , Mental Retardation, X-Linked/metabolism , Mice , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , RatsABSTRACT
The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT-/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT-/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT-/y mice. Similarly, CrT-/y neurons and P10 CrT-/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.
Subject(s)
Brain Diseases, Metabolic, Inborn/genetics , Brain/metabolism , Creatine/deficiency , DNA/genetics , Membrane Transport Proteins/genetics , Mental Retardation, X-Linked/genetics , Mutation , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Animals , Animals, Newborn , Brain/ultrastructure , Brain Diseases, Metabolic, Inborn/metabolism , Brain Diseases, Metabolic, Inborn/pathology , Creatine/genetics , Creatine/metabolism , DNA Mutational Analysis , Disease Models, Animal , Homeostasis , Male , Membrane Transport Proteins/deficiency , Mental Retardation, X-Linked/metabolism , Mental Retardation, X-Linked/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Microscopy, Electron , Neurons/metabolism , Neurons/ultrastructure , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolismABSTRACT
BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia. METHODS: RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo. RESULTS: SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3. CONCLUSIONS: Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.
Subject(s)
Creatine/metabolism , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Hypoxia/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Female , Heterografts , Humans , MCF-7 Cells , Mice , Mice, Nude , Middle Aged , Oxidative Stress , Triple Negative Breast Neoplasms/pathologyABSTRACT
The neurotransmitter:sodium symporter (NSS) homolog LeuT from Aquifex aeolicus has proven to be a valuable model for studying the transport mechanism of the NSS family. Crystal structures have captured LeuT in key conformations visited during the transport cycle, allowing for the construction of a nearly complete model of transport, with much of the conformational dynamics studied by computational simulations. Here, we report crystal structures of LeuT representing new intermediate conformations between the outward-facing open and occluded states. These structures, combined with binding and accessibility studies, reveal details of conformational dynamics that can follow substrate binding at the central substrate binding site (S1) of LeuT in outward-facing states, suggesting a potential competition for direction between the outward-open and outward-occluded states at this stage during substrate transport. Our structures further support an intimate interplay between the protonation state of Glu290 and binding of Na1 that may ultimately regulate the outward-open-to-occluded transition.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Sodium/metabolism , Aquifex/metabolism , Crystallography, X-Ray , Leucine/metabolism , Molecular Dynamics Simulation , Protein Conformation , Symporters/chemistry , Symporters/metabolism , ThermodynamicsABSTRACT
Creatine is an organic compound used as fast phosphate energy buffer to recycle ATP, important in tissues with high energy demand such as muscle or brain. Creatine is taken from the diet or endogenously synthetized by the enzymes AGAT and GAMT, and specifically taken up by the transporter SLC6A8. Deficit in the endogenous synthesis or in the transport leads to Cerebral Creatine Deficiency Syndromes (CCDS). CCDS are characterized by brain creatine deficiency, intellectual disability with severe speech delay, behavioral troubles such as attention deficits and/or autistic features, and epilepsy. Among CCDS, the X-linked creatine transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different mouse models of CTD were generated by doing long deletions in the Slc6a8 gene showing reduced brain creatine and cognitive deficiencies or impaired motor function. We present a new knock-in (KI) rat model of CTD holding an identical point mutation found in patients with reported lack of transporter activity. KI males showed brain creatine deficiency, increased urinary creatine/creatinine ratio, cognitive deficits and autistic-like traits. The Slc6a8Y389C KI rat fairly enriches the spectrum of CTD models and provides new data about the pathology, being the first animal model of CTD carrying a point mutation.
Subject(s)
Brain/metabolism , Nerve Tissue Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Animals , Base Sequence , Behavior, Animal , Body Weight , Brain Diseases, Metabolic, Inborn/genetics , Brain Diseases, Metabolic, Inborn/pathology , Creatine/blood , Creatine/deficiency , Creatine/genetics , Disease Models, Animal , Female , Gene Knock-In Techniques , Genotype , Humans , Male , Memory, Short-Term , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/pathology , Mutation, Missense , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , RatsSubject(s)
Brain Diseases, Metabolic, Inborn/diagnosis , Brain/metabolism , Creatine/deficiency , Creatine/metabolism , Mental Retardation, X-Linked/diagnosis , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Brain/diagnostic imaging , Brain Diseases, Metabolic, Inborn/diagnostic imaging , Brain Diseases, Metabolic, Inborn/metabolism , Child, Preschool , Humans , Magnetic Resonance Spectroscopy , Male , Mental Retardation, X-Linked/diagnostic imaging , Mental Retardation, X-Linked/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolismABSTRACT
Most therapeutic candidates for treating attention-deficit hyperactivity disorder (ADHD) have focused on modulating the dopaminergic neurotransmission system with neurotrophic factors. Regulation of this system by transcranial direct current stimulation (tDCS) could contribute to the recovery of cognitive symptoms observed in patients with ADHD. Here, male spontaneously hypertensive rats (SHR) were subjected to consecutive high-definition tDCS (HD-tDCS) (20 min, 50 µA, current density 63.7 A/m2, charge density 76.4 kC/m2) over the prefrontal cortex. This treatment alleviated cognitive deficits, with an increase in tyrosine hydroxylase and vesicular monoamine transporter two and significantly decreased plasma membrane reuptake transporter (DAT). HD-tDCS application increased the expression of several neurotrophic factors, particularly brain-derived neurotrophic factor (BDNF), and activated hippocampal neurogenesis. Our results suggest that anodal HD-tDCS over the prefrontal cortex may ameliorate cognitive dysfunction via regulation of DAT and BDNF in the mesocorticolimbic dopaminergic pathways, and therefore represents a potential adjuvant therapy for ADHD.
Subject(s)
Attention Deficit Disorder with Hyperactivity/metabolism , Prefrontal Cortex , Transcranial Direct Current Stimulation , Animals , Disease Models, Animal , Male , Nerve Growth Factors/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Prefrontal Cortex/chemistry , Prefrontal Cortex/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKYABSTRACT
BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) have intestinal barrier dysfunction. Creatine regulates energy distribution within cells and reduces the severity of colitis in mice. We studied the functions of the creatine transporter solute carrier family 6 member 8 (SLC6A8, also called CRT) in intestinal epithelial cells (IECs) and mice, and we measured levels in mucosal biopsies from patients with IBD. METHODS: Colon biopsy specimens from patients with IBD (30 with Crohn's disease and 27 with ulcerative colitis) and 30 patients without IBD (control individuals) and colon tissues from mice (with and without disruption of Crt) were analyzed by immunofluorescence, immunoblots, and/or quantitative reverse-transcription polymerase chain reaction (qRT-PCR). CRT was knocked down or overexpressed in T84 cells, which were analyzed by immunofluorescence, immunoblots, high-performance liquid chromatography (to measure creatine levels), qRT-PCR, transepithelial electrical resistance, barrier function, actin localization, wound healing, mitochondrial oxygen consumption, and glycolysis extracellular acidification rate assays. Organoids from colon cells of CRT-knockout mice and control mice were analyzed by qRT-PCR, immunoblot, and transepithelial electrical resistance. RESULTS: CRT localized around tight junctions (TJs) of T84 IECs. In analyses of IECs with CRT knockdown or overexpression, we found that CRT regulates intracellular creatine, barrier formation, and wound healing. CRT-knockout organoids also had diminished barrier formation. In the absence of adequate creatine, IECs transition toward a stressed, glycolysis-predominant form of metabolism; this resulted in leaky TJs and mislocalization of actin and TJ proteins. Colon tissues from patients with IBD had reduced levels of CRT messenger RNA compared with those from control individuals. CONCLUSIONS: In an analysis of IEC cell lines and colonoids derived from CRT-knockout mice, we found that CRT regulates energy balance in IECs and thereby epithelial integrity and barrier function. Mucosal biopsy specimens from patients with ulcerative colitis and inactive Crohn's disease have lower levels of CRT, which might contribute to the reduced barrier function observed in patients with IBD.
Subject(s)
Colitis, Ulcerative/pathology , Colon/pathology , Crohn Disease/pathology , Intestinal Mucosa/pathology , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Adult , Animals , Biopsy , Case-Control Studies , Cell Line , Energy Metabolism , Epithelial Cells/cytology , Epithelial Cells/pathology , Female , Gene Knockdown Techniques , Humans , Male , Membrane Transport Proteins/genetics , Mice , Mice, Knockout , Middle Aged , Mitochondria/metabolism , Nerve Tissue Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Tight Junctions/pathologyABSTRACT
Creatine is a crucial metabolite that plays a fundamental role in ATP homeostasis in tissues with high-energy demands. The creatine transporter (CreaT, SLC6A8) belongs to the solute carrier 6 (SLC6) transporters family, and more particularly to the GABA transporters (GATs) subfamily. Understanding the molecular determinants of specificity within the SLC6 transporters in general, and the GATs in particular is very challenging due to the high similarity of these proteins. In the study presented here, our efforts focused on finding key structural features involved in binding selectivity for CreaT using structure-based computational methods. Due to the lack of three-dimensional structures of SLC6A8, our approach was based on the realization of two reliable homology models of CreaT using the structures of two templates, i.e. the human serotonin transporter (hSERT) and the prokaryotic leucine transporter (LeuT). Our models reveal that an optimal complementarity between the shape of the binding site and the size of the ligands is necessary for transport. These findings provide a framework for a deeper understanding of substrate selectivity of the SLC6 family and other LeuT fold transporters.
Subject(s)
Creatine/metabolism , Molecular Docking Simulation , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Aquifex , Bacterial Proteins/ultrastructure , Binding Sites , Creatine/chemistry , Ligands , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/ultrastructure , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/ultrastructure , Protein Conformation, alpha-Helical , Sequence Alignment , Sequence Homology, Amino Acid , Serotonin Plasma Membrane Transport Proteins/ultrastructure , Substrate SpecificityABSTRACT
More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.
Subject(s)
Brain Diseases, Metabolic, Inborn/metabolism , Creatine/deficiency , Mental Retardation, X-Linked/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Brain Diseases, Metabolic, Inborn/genetics , Creatine/genetics , Creatine/metabolism , HEK293 Cells , Humans , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mental Retardation, X-Linked/genetics , Mutation, Missense , Nerve Tissue Proteins/chemistry , Phenylbutyrates/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolismABSTRACT
Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Leucine/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Amino Acid Substitution , Aquifex , Bacteria/chemistry , Bacteria/genetics , Bacteria/metabolism , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Protein ConformationABSTRACT
Secondary active transporters use electrochemical gradient of ions to fuel the "uphill" translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF â IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF â IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.
Subject(s)
Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Sodium/metabolismABSTRACT
Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.