Jessica Johansson

Altered tryptophan and alanine transport in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD) : an in vitro study

BackgroundThe catecholaminergic and serotonergic neurotransmitter systems are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD). The amino acid tyrosine is the precursor for synthesis of the catecholamines dopamine and norepinephrine, while tryptophan is the precursor of serotonin. A disturbed transport of tyrosine, as well as other amino acids, has been found in a number of other psychiatric disorders, such as schizophrenia, bipolar disorder and autism, when using the fibroblast cell model. Hence, the aim of this study was to explore whether children with ADHD may have disturbed amino acid transport.MethodsFibroblast cells were cultured from skin biopsies obtained from 14 boys diagnosed with ADHD and from 13 matching boys without a diagnosis of a developmental disorder. Transport of the amino acids tyrosine, tryptophan and alanine across the cell membrane was measured by the cluster tray method. The kinetic parameters, maximal transport capacity (Vmax ) and affinity constant (Km ) were determined. Any difference between the two groups was analyzed by Students unpaired t-test or the Mann Whitney U test.ResultsThe ADHD group had significantly decreased Vmax (p = 0.039) and Km (increased affinity) (p = 0.010) of tryptophan transport in comparison to controls. They also had a significantly higher Vmax of alanine transport (p = 0.031), but the Km of alanine transport did not differ significantly. There were no significant differences in any of the kinetic parameters regarding tyrosine transport in fibroblasts for the ADHD group.ConclusionsTryptophan uses the same transport systems in both fibroblasts and at the blood brain barrier (BBB). Hence, a decreased transport capacity of tryptophan implies that less tryptophan is being transported across the BBB in the ADHD group. This could lead to deficient serotonin access in the brain that might cause disturbances in both the serotonergic and the catecholaminergic neurotransmitter systems, since these systems are highly interconnected. The physiological importance of an elevated transport capacity of alanine to the brain is not known to date.

Aberrant amino acid transport in fibroblasts from patients with bipolar disorder

Aberrant tyrosine transport is a repeated finding in fibroblasts from schizophrenic patients. The transport aberration could lead to disturbances in the dopaminergic and noradrenergic neurotransmitter systems. Tyrosine and tryptophan are the precursors of the neurotransmitters dopamine and serotonin. Disturbed dopaminergic, noradrenergic and serotoninergic systems are implicated as causes of bipolar disorder. Hence, the aim of this study was to explore whether patients with bipolar disorder have an aberrant transport of tyrosine and/or tryptophan. Fibroblast cell lines from patients with bipolar type-1 disorder (n=10) and healthy controls (n=10) were included in this study. All patients fulfilled the DSM-IV diagnostic criteria. The transport of amino acids across the cell membranes was measured by the cluster tray method. The kinetic parameters, maximal transport velocity (V(max)) and affinity constant (K(m)) were determined. A significantly lower V(max) for tyrosine (p=0.027) was found in patients with bipolar type-1 disorder in comparison to healthy controls. No significant differences in K(m) for tyrosine and in the kinetic parameters of tryptophan between patients with bipolar type-1 disorder and healthy controls were observed. The decreased tyrosine transport (low V(max)) found in this study may indicate less access of dopamine in the brain, resulting in disturbed dopaminergic and/or noradrenergic neurotransmission, that secondarily could lead to disturbances in other central neurotransmitter systems, such as the serotoninergic system. However, as sample size was small in this study and an age difference between patients and controls existed, the present findings should be considered as pilot data. Further studies with larger sample number are needed to elucidate the transport aberration and the significance of these findings.

Tryptophan Transport in Human Fibroblast Cells— A Functional Characterization

There are indications that serotonergic neurotransmission is disturbed in several psychiatric disorders. One explanation may be disturbed transport of tryptophan (precursor for serotonin synthesis) across cell membranes. Human fibroblast cells offer an advantageous model to study the transport of amino acids across cell membranes, since they are easy to propagate and the environmental factors can be controlled. The aim of this study was to functionally characterize tryptophan transport and to identify the main transporters of tryptophan in fibroblast cell lines from healthy controls. Tryptophan kinetic parameters (Vmax and Km) at low and high concentrations were measured in fibroblasts using the cluster tray method. Uptake of 3H (5)-L-tryptophan at different concentrations in the presence and absence of excess concentrations of inhibitors or combinations of inhibitors of amino acid transporters were also measured. Tryptophan transport at high concentration (0.5 mM) had low affinity and high Vmax and the LAT1 isoform of system-L was responsible for approximately 40% of the total uptake of tryptophan. In comparison, tryptophan transport at low concentration (50 nM) had higher affinity, lower Vmax and approximately 80% of tryptophan uptake was transported by system-L with LAT1 as the major isoform. The uptake of tryptophan at the low concentration was mainly sodium (Na+) dependent, while uptake at high substrate concentration was mainly Na+ independent. A series of different transporter inhibitors had varying inhibitory effects on tryptophan uptake. This study indicates that tryptophan is transported by multiple transporters that are active at different substrate concentrations in human fibroblast cells. The tryptophan transport trough system-L was mainly facilitated by the LAT1 isoform, at both low and high substrate concentrations of tryptophan.

Genetic and Functional Study of L-Type Amino Acid Transporter 1 in Schizophrenia.

Schizophrenia involves neural catecholaminergic dysregulation. Tyrosine is the precursor of catecholamines, and its major transporter, according to studies on fibroblasts, in the brain is the L-type amino acid transporter 1 (LAT1). The present study assessed haplotype tag single-nucleotide polymorphisms (SNPs) of the SLC7A5/LAT1 gene in 315 patients with psychosis within the schizophrenia spectrum and 233 healthy controls to investigate genetic vulnerability to the disorder as well as genetic relationships to homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG), the major catecholamine metabolites in the cerebrospinal fluid (CSF). Moreover, the involvement of the different isoforms of the system L in tyrosine uptake and LAT1 tyrosine kinetics were studied in fibroblast cell lines of 10 patients with schizophrenia and 10 healthy controls. The results provide suggestive evidence of individual vulnerability to schizophrenia related to the LAT1 SNP rs9936204 genotype. A number of SNPs were nominally associated with CSF HVA and MHPG concentrations but did not survive correction for multiple testing. The LAT1 isoform was confirmed as the major tyrosine transporter in patients with schizophrenia. However, the kinetic parameters (maximal transport capacity, affinity of the binding sites, and diffusion constant of tyrosine transport through the LAT1 isoform) did not differ between patients with schizophrenia and controls. The present genetic findings call for independent replication in larger samples, while the functional study seems to exclude a role of LAT1 in the aberrant transport of tyrosine in fibroblasts of patients with schizophrenia.

Proinflammatory cytokines and oxidative stress decrease the transport of dopamine precursor tyrosine in human fibroblasts

Background: Proinflammatory cytokines and oxidative stress responses have been extensively implicated in the pathophysiology of neuropsychiatric disorders over the past 2 decades. Moreover, disturbed transport of the dopamine precursor (i.e., the amino acid tyrosine) has been demonstrated, in different studies, across fibroblast cell membranes obtained from neuropsychiatric patients. However, the role and influences of proinflammatory cytokines and oxidative stress, and the reasons for disturbed tyrosine transport in neuropsychiatric disorders, are still not evaluated. Aims: The present study aimed to assess the role of proinflammatory cytokines and oxidative stress, indicated in many neuropsychiatric disorders, in tyrosine transportation, by using human skin-derived fibroblasts. Methods: Fibroblasts obtained from a healthy control were used in this study. Fibroblasts were treated with proinflammatory cytokines (IL-1β, IFN-γ, IL-6, TNF-α), their combinations, and oxidative stress, optimized for concentrations and incubation time, to analyze the uptake of 14C-tyrosine compared to untreated controls. Results and Conclusion: This study demonstrates that proinflammatory cytokines and oxidative stress decrease the transport of tyrosine (47% and 33%, respectively), which can alter dopamine synthesis. The functionality of the tyrosine transporter could be a new potential biomarker to target for discovering new drugs to counteract the effects of proinflammatory cytokines and oxidative stress in the pathophysiology of neuropsychiatric disorders.