Transporter Deficiency Syndrome Research Articles

Breaking the rules of SLC6 transporters: Export of the human creatine transporter-1 from the endoplasmic reticulum is supported by its N-terminus.

Mutations in the human creatine transporter 1 (CRT1/SLC6A8) cause the creatine transporter deficiency syndrome, which is characterized by intellectual disability, epilepsy, autism, and developmental delay. The vast majority of mutations cause protein misfolding and hence reduce cell surface expression. Hence, it is important to understand the molecular machinery supporting folding and export of CRT1 from the endoplasmic reticulum (ER). All other SLC6 members thus far investigated rely on a C-terminal motif for binding the COPII-component SEC24 to drive their ER export; their N-termini are dispensable. Here, we show that, in contrast, in CRT1 the C-terminal ER-export motif is cryptic and it is the N-terminus, which supports ER export. This conclusion is based on the following observations: (i) siRNA-induced depletion of individual SEC24 isoforms revealed that CRT1 relied on SEC24C for ER export. However, mutations of the C-terminal canonical ER-export motif of CRT1 did not impair its cell surface delivery. (ii) Nevertheless, the C-terminal motif of CRT1 was operational in a chimeric protein comprising the serotonin transporter (SERT/SLC6A4) and the C-terminus of CRT1. (iii) Tagging of the N-terminus-but not the C-terminus-with yellow fluorescent protein (YFP) resulted in ER retention. (iv) Serial truncations of the N-terminus showed that removal of ≥51 residues of CRT1 impaired surface delivery, because the truncated CRT1 were confined to the ER. (v) Mutation of P51 to alanine also reduced cell surface delivery of CRT1 and relieved its dependence on SEC24C. Thus, the ER-export motif in the N-terminus of CRT1 overrides the canonical C-terminal motif.

Probing binding and occlusion of substrate in the human creatine transporter-1 by computation and mutagenesis.

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane-embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148-creatine-Na+ is essential to the process of occlusion, which occurs via a "hold-and-pull" mechanism. The model should be useful to study the impact of disease-associated point mutations on the folding of CRT1 and identify approaches which correct folding-deficient mutants.

Dopamine Transporter Deficiency Syndrome (DTDS): Expanding the Clinical Phenotype and Precision Medicine Approaches.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

Gene Therapy for Dopamine Dyshomeostasis: From Parkinson's to Primary Neurotransmitter Diseases.

Neurological disorders encompass a broad range of neurodegenerative and neurodevelopmental diseases that are complex and almost universally without disease modifying treatments. There is, therefore, significant unmet clinical need to develop novel therapeutic strategies for these patients. Viral gene therapies are a promising approach, where gene delivery is achieved through viral vectors such as adeno-associated virus and lentivirus. The clinical efficacy of such gene therapies has already been observed in two neurological disorders of pediatric onset; for spinal muscular atrophy and aromatic L-amino acid decarboxylase (AADC) deficiency, gene therapy has significantly modified the natural history of disease in these life-limiting neurological disorders. Here, we review recent advances in gene therapy, focused on the targeted delivery of dopaminergic genes for Parkinson's disease and the primary neurotransmitter disorders, AADC deficiency and dopamine transporter deficiency syndrome (DTDS). Although recent European Medicines Agency and Medicines and Healthcare products Regulatory Agency approval of Upstaza (eladocagene exuparvovec) signifies an important landmark, numerous challenges remain. Future research will need to focus on defining the optimal therapeutic window for clinical intervention, better understanding of the duration of therapeutic efficacy, and improved brain targeting. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

One Molecule for Mental Nourishment and More: Glucose Transporter Type 1-Biology and Deficiency Syndrome.

Glucose transporter type 1 (Glut1) is the main transporter involved in the cellular uptake of glucose into many tissues, and is highly expressed in the brain and in erythrocytes. Glut1 deficiency syndrome is caused mainly by mutations of the SLC2A1 gene, impairing passive glucose transport across the blood–brain barrier. All age groups, from infants to adults, may be affected, with age-specific symptoms. In its classic form, the syndrome presents as an early-onset drug-resistant metabolic epileptic encephalopathy with a complex movement disorder and developmental delay. In later-onset forms, complex motor disorder predominates, with dystonia, ataxia, chorea or spasticity, often triggered by fasting. Diagnosis is confirmed by hypoglycorrhachia (below 45 mg/dL) with normal blood glucose, 18F-fluorodeoxyglucose positron emission tomography, and genetic analysis showing pathogenic SLC2A1 variants. There are also ongoing positive studies on erythrocytes’ Glut1 surface expression using flow cytometry. The standard treatment still consists of ketogenic therapies supplying ketones as alternative brain fuel. Anaplerotic substances may provide alternative energy sources. Understanding the complex interactions of Glut1 with other tissues, its signaling function for brain angiogenesis and gliosis, and the complex regulation of glucose transportation, including compensatory mechanisms in different tissues, will hopefully advance therapy. Ongoing research for future interventions is focusing on small molecules to restore Glut1, metabolic stimulation, and SLC2A1 transfer strategies. Newborn screening, early identification and treatment could minimize the neurodevelopmental disease consequences. Furthermore, understanding Glut1 relative deficiency or inhibition in inflammation, neurodegenerative disorders, and viral infections including COVID-19 and other settings could provide clues for future therapeutic approaches.

Structure-Activity Relationships of Dopamine Transporter Pharmacological Chaperones.

Mutations in the dopamine transporter gene (SLC6A3) have been implicated in many human diseases. Among these is the infantile parkinsonism-dystonia known as Dopamine Transporter Deficiency Syndrome (DTDS). Afflicted individuals have minimal to no functional dopamine transporter protein. This is primarily due to retention of misfolded disease-causing dopamine transporter variants. This results in a variety of severe motor symptoms in patients and the disease ultimately leads to death in adolescence or young adulthood. Though no treatment is currently available, pharmacological chaperones targeting the dopamine transporter have been shown to rescue select DTDS disease-causing variants. Previous work has identified two DAT pharmacological chaperones with moderate potency and efficacy: bupropion and ibogaine. In this study, we carried out structure-activity relationships (SARs) for bupropion and ibogaine with the goal of identifying the chemical features required for pharmacological chaperone activity. Our results show that the isoquinuclidine substituent of ibogaine and its analogs is an important feature for pharmacological chaperone efficacy. For bupropion, the secondary amine group is essential for pharmacological chaperone activity. Lastly, we describe additional ibogaine and bupropion analogs with varying chemical modifications and variable pharmacological chaperone efficacies at the dopamine transporter. Our results contribute to the design and refinement of future dopamine transporter pharmacological chaperones with improved efficacies and potencies.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

Characterization of Speech and Language Phenotype in GLUT1DS

Background: To analyze the oral motor, speech and language phenotype in a sample of pediatric patients with GLUT 1 transporter deficiency syndrome (GLUT1DS). Methods: eight Italian-speaking children with GLUT1DS (aged 4.6–15.4 years) in stable treatment with ketogenic diet from a variable time underwent a specific and standardized speech and language assessment battery. Results: All patients showed deficits with different degrees of impairment in multiple speech and language areas. In particular, orofacial praxis, parallel and total movements were the most impaired in the oromotor domain; in the speech domain patients obtained a poor performance in the diadochokinesis rate and in the repetition of words that resulted as severely deficient in seven out of eight patients; in the language domain the most affected abilities were semantic/phonological fluency and receptive grammar. Conclusions: GLUT1DS is associated to different levels of speech and language impairment, which should guide diagnostic and therapeutic intervention. Larger population data are needed to identify more precisely a speech and language profile in GLUT1DS patients.

PRO10 Identifying Appropriate PROXY Diseases for Estimating LONG-TERM Survival and IMPACT on Health-Related Quality of Life of Patients with Aromatic L-Amino Acid Decarboxylase Deficiency (AADC-D)

We aimed to identify diseases similar in characteristics to AADC deficiency (AADC-d) that can provide proxy estimates for health-related quality of life (HRQoL) and long-term survival of AADC-d patients. These estimates will be used to support the economic model development for eladocagene exuparvovec. A literature search and clinician survey were conducted to identify proxy diseases for AADC-d. The literature was searched for observational studies, single arm clinical trials, randomized clinical trials, systematic reviews, case studies and case reports between January 2015 and January 2020 from clinicaltrials.gov and PubMed.gov. Studies among paediatric patients with motor associated neurological, genetic, or inherited conditions were included. Additionally, a survey was administered electronically to 25 clinicians globally to assess appropriateness of anoxic encephalopathy, paraplegia, tyrosine hydroxylase (TH) deficiency, guanosine triphosphate cyclohydrolase deficiency, sepiapterin reductase deficiency (SR), dopamine transporter deficiency syndrome as proxies. Appropriateness of disease proxy was determined based on similarity in disease characteristics, disease onset, natural history of disease progression, and data availability. Spinal muscular atrophy (SMA), Duchenne muscular dystrophy, metachromatic leukodystrophy, and cerebral palsy (CP) were identified in the literature search as suitable proxies. Survival and HRQoL data were available for SMA type I and CP since motor specific developmental milestones were measured as an endpoint in the related clinical trials. Survey results indicate TH deficiency and SR deficiency could be suitable proxies, but a lack of published data on survival and HRQoL limit their use for economic modeling in AADC-d. CP was preferred by clinicians as an appropriate proxy in AADC-d, in the absence of required data for other proxies. This study provides evidence to fill a gap in the literature for AADC-d patients’ outcomes. The available mortality and HRQoL data for the proxy diseases may be used to estimate the long-term outcomes for patients with AADC-d.

Expanding the phenotypic spectrum of dopamine transporter deficiency syndrome with a novel mutation (1884)

Expanding the phenotypic spectrum of dopamine transporter deficiency syndrome with a novel mutation (1884)

Review of Riboflavin Transporter Deficiency Syndrome: Sameness in Genotype occurs with Variations in Phenotype.

Riboflavin Transporter Deficiency Syndrome (RTD) is a rare childhood neurodegenerative disorder. RTD is acquired as an autosomal recessive pattern, which indicates that a child whose parents are carriers of RTD has a 25% chance of having the disease [homozygous for the RTD trait], a 50% chance of being a carrier [a heterozygous for the RTD trait], and a 25% chance of not having the disease [homozygous negative for the RTD trait]. RTD is a sensory motor neuron disease with symptoms such as auditory neuropathy, sensory ataxia, sensorineural deafness, and muscle weakness. These symptoms were first described in 1892 but diagnosis only became established in 2009 following the application of molecular biology investigation to discern the cause. Through exome sequencing and genetic testing, a mutation of the Solute Carrier 52A gene (SLC52A) was found to be implicated in RTD. There is no cure for RTD currently, but dietary Riboflavin supplementation has been shown to be a very effective treatment for most RTD patients. The current treatment is based on mg Riboflavin/kg of Body Weight/day. This approach fails to consider the role of other potential factors in the efficacy of the therapy, as well as in the variations and severity of the symptoms. This research uses case reports and meta‐analysis of available work reports to highlight the potential pathophysiological factors that may contribute to the variations, severity of signs and symptoms reported, and treatment responses in RTD affected patients. This report includes the molecular, cellular, and the phenotypic dynamics of RTD, and ends with a review of reported treatment responses. It is believed that such a hierarchical assessment will better equip the patients, their families, and the attending clinicians with the knowledge to assess and treat each RTD patient individually and with greater accuracy, success and outcomes than in the past.Support or Funding InformationThis research supported by Institutional Resources of the University of Science Arts and Technology, Montserrat, British West Indies and the Einstein Institute, USA.

Homozygous in-frame variant of SCL6A3 causes dopamine transporter deficiency syndrome in a consanguineous family.

The human dopamine transporter (hDAT) participates in dopamine homeostasis by clearing dopamine from the extracellular space using secondary active transport. Dysregulation of hDAT has been reported to be associated with different neuropsychiatric disorders. Dopamine transporter deficiency syndrome (DTDS) is a complex disease caused by defects in dopamine uptake within the synaptic cleft and patients manifest parkinsonian features. The extracellular loops are crucial for DAT activity and defects in these regions disturb dopamine transport. In the present study, a 3.5-year-old female in a consanguineous Iranian family with an initial diagnosis of gait imbalance and speech delay has been identified. We utilized whole-exome sequencing (WES) to identify the possible genetic defect(s). WES identified a novel homozygous in-frame indel variant, c.1139_1150del; p.(Gly380_Lys384delinsGlu), in the SLC6A3 gene (NM_001044.4), as the most likely disease-susceptibility variant. This variant is located in extracellular loop 4 (EL4) of the DAT protein. Our study highlights the role of extracellular loops and shows the EL4 of hDAT as a critical region for the protein activity. The identified variant in the EL4 region of DAT is predicted to compromise DAT function and may lead to DTDS in this case. However, complementary studies are required to confirm.

Epilepsy in de Vivo syndrome: a literature review and a clinical case

GLUT 1 transporter deficiency syndrome (De Vivo syndrome, GLUT 1 deficiency syndrome, De Vivo Disease) is a rare genetic disease associated with a deficiency of the glucose transporter GLUT 1. Due to this deficiency, diffusion of glucose through the blood-brain barrier is limited or completely blocked. As a result, a clinical symptom complex of neurological disorders – epileptic seizures, cognitive deficit, and motor disorders – develops. The article provides a review of the literature on the variety of epilepsy manifestations in this syndrome, including idiopathic generalized epilepsy with absences, myoclonic-astatic epilepsy, and focal epilepsy. The inability of the basic antiepileptic therapy to stop the seizures is noted. The main treatment method is the ketogenic diet. A clinical observation made by these authors is also presented.

Juvenile onset parkinsonism-dystonia syndrome: A novel mutation in the SLC6A3 gene

The differential diagnosis for genetic dystonia parkinsonism is broad. Juvenile onset parkinsonism-dystonia, also known as dopamine transporter deficiency syndrome (DTDS), is an autosomal recessive disorder with variable neurological manifestations and age of onset. We describe DTDS with a novel mutation. Case report We identified a 19-year-old male, who presented with history of juvenile-onset parkinsonism. He had a normal birth and neonatal course with normal developmental milestones. Cognitive, motor and speech were reported to be normal in the first decade of life. At 10–11 years of age, the patient started to have tremor and progressive parkinsonian symptoms. He additionally starts to have speech difficulties. The parents are consanguineous, but there is no family history of movement disorders, Parkinson’s disease or other psychiatric illness. Other siblings (brother and sister) are unaffected. Neurological examination revealed loss of speech to single words, rigidity, generalized bradykinesia, dystonia with dystonic tremor. Gene analysis showed a novel homozygous mutation in SLC6A3 gene (g.1394863c > G) with both parents and siblings having a heterozygous mutation in the same gene. •DTDS is a rare disorder that should be considered in the differential diagnosis of genetic parkinsonism of juvenile-onset. Affected individuals show hyperkinesia with orolingual and limb dyskinesia, dystonia, and chorea with parkinsonian features.•Atypical onset in adolescence or adulthood has been described.•SLC6A3 encodes the dopamine transporter (DAT) that is expressed predominantly within the substantia nigra and in the midbrain, which has a crucial role in mediating reuptake and homeostasis of dopamine.

Glucose transporter deficienc y syndrome type 1: a case report

The authors present a unique clinical observation of the case of a hereditary deficiency of a type 1 glucose transporter, also called de Vivo disease. The type 1 glucose transporter deficiency syndrome (OMIM: 606777, ORPHA: 71277) is an extremely rare genetic disease associated with mutations in the SCL2A gene encoding the transfer of glucose across the blood-brain barrier. The clinical case, given in the article, replenishes the piggy bank of genetically verified glucose transport disruption syndromes leading to the development of polymorphic neurological disorders. The syndrome is most often inherited by an autosomal dominant pathway, but rare cases of autosomal recessive transmission are known. About 500 clinical cases of the present syndrome are described, although, according to various authors, the number of clinically verified cases not confirmed by genetic verification is much higher. In addition to the given data of the clinical course of the disease, detailed results of the genetic interpretation of the hereditary syndrome are given, the modern method of pathogenetic therapy is considered – the ketogenic diet.

4‐Phenylbutyrate rescues folding‐deficient creatine transporter‐1 variants linked to the creatine transporter deficiency syndrome

Mutations in the coding sequence of the hCRT‐1 gene (SLC6A8) have been associated with the creatine transporter deficiency (CTD) syndrome. CTD encompasses a range of moderate to severe conditions, from epilepsy, mental retardation, autism, development delay, behavioural problems and motor dysfunction to gastrointestinal symptoms. Diseases arising from misfolding of other SLC6 transporters have been reported over recent years; e.g. folding‐deficient mutants of the dopamine transporter (hDAT) cause infantile/juvenile Parkinsonism dystonia in children, while orthostatic intolerance arises from misfolding of the human noradrenaline transporter (hNET). In hCRT‐1, dozens of naturally occurring variants have been linked to CTD. Remarkably, some of these mutations occur at highly conserved residues across the SLC6 family; e.g. a single point mutation inducing a sleepless phenotype in Drosophila DAT (dDAT‐G108Q), leads to severe mental retardation in children when mutated at the equivalent glycine residue in hCRT‐1 (hCRT‐1‐G132V). Similarly, the P554L mutation causes Parkinsonism/dystonia in hDAT, but triggers drug‐resistant epilepsy in children harbouring the same mutation in hCRT‐1. Here, we examined 16 clinically relevant hCRT‐1 variants to assess the molecular basis of CTD. Confocal microscopy imaging revealed that the YFP‐tagged mutant CRTs are trapped in the endoplasmic reticulum (ER), co‐localised with an ER‐resident chaperone calnexin, whereas the wild type hCRT‐1 reached the plasma membrane. The chemical chaperone 4‐phenylbutyrate (4‐PBA) proved to be effective in functionally rescuing several of the folding‐deficient CRT‐1 mutants. Radiolabeled creatine uptake assays were also verified by assessing the surface expression of the variant transporters by cell surface biotinylation and immunoblotting of detergent lysates (i.e. the mature fully‐glycosylated band appeared in response to positive chaperone action). In addition, the rescue action of 4‐PBA was also recapped in primary hippocampal neurons expressing the wild type transporter and CTD‐triggering mutations. Overall, this work is clinically relevant and ought to confer pristine therapeutic prospects awaited for by many CTD patients.Support or Funding InformationThis work was supported by the Austrian Science Fund (FWF) project no P31255‐B27 to Sonja Sucic.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rescue by 4-phenylbutyrate of several misfolded creatine transporter-1 variants linked to the creatine transporter deficiency syndrome

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials.This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake.

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson’s disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

Individualizing Treatment Approaches for Epileptic Patients with Glucose Transporter Type1 (GLUT-1) Deficiency.

Monogenic and polygenic mutations are important contributors in patients suffering from epilepsy, including metabolic epilepsies which are inborn errors of metabolism with a good respond to specific dietetic treatments. Heterozygous variation in solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1) and mutations of the GLUT1/SLC2A2 gene results in the failure of glucose transport, which is related with a glucose type-1 transporter (GLUT1) deficiency syndrome (GLUT1DS). GLUT1 deficiency syndrome is a treatable disorder of glucose transport into the brain caused by a variety of mutations in the SLC2A1 gene which are the cause of different neurological disorders also with different types of epilepsy and related clinical phenotypes. Since patients continue to experience seizures due to a pharmacoresistance, an early clinical diagnosis associated with specific genetic testing in SLC2A1 pathogenic variants in clinical phenotypes could predict pure drug response and might improve safety and efficacy of treatment with the initiation of an alternative energy source including ketogenic or analog diets in such patients providing individualized strategy approaches.