RESUMEN
The antidepressants trazodone and nefazodone were approved some 4 and 3 decades ago, respectively. Their action is thought to be mediated, at least in part, by inhibition of the serotonin transporter [SERT/solute carrier (SLC)-6A4]. Surprisingly, their mode of action on SERT has not been characterized. Here, we show that, similar to the chemically related drug vilazodone, trazodone and nefazodone are allosteric ligands: trazodone and nefazodone inhibit uptake by and transport-associated currents through SERT in a mixed-competitive and noncompetitive manner, respectively. Contrary to noribogaine and its congeners, all three compounds preferentially interact with the Na+-bound outward-facing state of SERT. Nevertheless, they act as pharmacochaperones and rescue the folding-deficient variant SERT-P601A/G602A. The vast majority of disease-associated point mutations of SLC6 family members impair folding of the encoded transporter proteins. Our findings indicate that their folding defect can be remedied by targeting allosteric sites on SLC6 transporters. SIGNIFICANCE STATEMENT: The serotonin transporter is a member of the solute carrier-6 family and is the target of numerous antidepressants. Trazodone and nefazodone have long been used as antidepressants. Here, this study shows that their inhibition of the serotonin transporter digressed from the competitive mode seen with other antidepressants. Trazodone and nefazodone rescued a folding-deficient variant of the serotonin transporter. This finding demonstrates that folding defects of mutated solute carrier-6 family members can also be corrected by allosteric ligands.
Asunto(s)
Antidepresivos , Piperazinas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Trazodona , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Trazodona/farmacología , Trazodona/metabolismo , Humanos , Antidepresivos/farmacología , Antidepresivos/metabolismo , Piperazinas/farmacología , Piperazinas/metabolismo , Regulación Alostérica/efectos de los fármacos , Células HEK293 , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Triazoles/farmacología , Pliegue de Proteína/efectos de los fármacos , Clorhidrato de Vilazodona/farmacología , Clorhidrato de Vilazodona/metabolismoRESUMEN
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.
RESUMEN
The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.
Asunto(s)
Proteínas de Transporte de Serotonina en la Membrana Plasmática , Serotonina , Humanos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Serotonina/metabolismo , Células HEK293 , Transporte IónicoRESUMEN
The human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) is the first member of the solute carrier 6 (SLC6) protein superfamily. GAT-1 (SLC6A1) is one of the main GABA transporters in the central nervous system. Its principal physiological role is retrieving GABA from the synapse into neurons and astrocytes, thus swiftly terminating neurotransmission. GABA is a key inhibitory neurotransmitter and shifts in GABAergic signaling can lead to pathological conditions, from anxiety and epileptic seizures to schizophrenia. Point mutations in the SLC6A1 gene frequently give rise to epilepsy, intellectual disability or autism spectrum disorders in the afflicted individuals. The mechanistic routes underlying these are still fairly unclear. Some loss-of-function variants impair the folding and intracellular trafficking of the protein (thus retaining the transporter in the endoplasmic reticulum compartment), whereas others, despite managing to reach their bona fide site of action at the cell surface, nonetheless abolish GABA transport activity (plausibly owing to structural/conformational defects). Whatever the molecular culprit(s), the physiological aftermath transpires into the absence of functional transporters, which in turn perturbs GABAergic actions. Dozens of mutations in the kin SLC6 family members are known to exhort protein misfolding. Such events typically elicit severe ailments in people, e.g., infantile parkinsonism-dystonia or X-linked intellectual disability, in the case of dopamine and creatine transporters, respectively. Flaws in protein folding can be rectified by small molecules known as pharmacological and/or chemical chaperones. The search for such apt remedies calls for a systematic investigation and categorization of the numerous disease-linked variants, by biochemical and pharmacological means in vitro (in cell lines and primary neuronal cultures) and in vivo (in animal models). We here give special emphasis to the utilization of the fruit fly Drosophila melanogaster as a versatile model in GAT-1-related studies. Jointly, these approaches can portray indispensable insights into the molecular factors underlying epilepsy, and ultimately pave the way for contriving efficacious therapeutic options for patients harboring pathogenic mutations in hGAT-1.
RESUMEN
Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. We systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. We here focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. We employed diverse small molecules to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). We identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. Our data grant crucial new insights into: (i) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (ii) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.
RESUMEN
Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG584,585AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.
RESUMEN
Neurodegenerative disorders (ND) like Alzheimer's (AD), Parkinson's (PD), Huntington's or Prion diseases share similar pathological features. They are all age dependent and are often associated with disruptions in analogous metabolic processes such as protein aggregation and oxidative stress, both of which involve metal ions like copper, manganese and iron. Bush and Tanzi proposed 2008 in the 'metal hypothesis of Alzheimer's disease' that a breakdown in metal homeostasis is the main cause of NDs, and drugs restoring metal homeostasis are promising novel therapeutic strategies. We report here that metallothionein (MT), an endogenous metal detoxifying protein, is increased in young amyloid ß (Aß) expressing Caenorhabditis elegans, whereas it is not in wild type strains. Further MT induction collapsed in 8 days old transgenic worms, indicating the age dependency of disease outbreak, and sharing intriguing parallels to diminished MT levels in human brains of AD. A medium throughput screening assay method was established to search for compounds increasing the MT level. Compounds known to induce MT release like progesterone, ZnSO4, quercetin, dexamethasone and apomorphine were active in models of AD and PD. Thioflavin T, clioquinol and emodin are promising leads in AD and PD research, whose mode of action has not been fully established yet. In this study, we could show that the reduction of Aß and α-synuclein toxicity in transgenic C. elegans models correlated with the prolongation of MT induction time and that knockdown of MT with RNA interference resulted in a loss of bioactivity.
Asunto(s)
Envejecimiento/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Metalotioneína/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Animales Modificados Genéticamente , Benzotiazoles/administración & dosificación , Benzotiazoles/farmacología , Clioquinol/administración & dosificación , Clioquinol/farmacología , Modelos Animales de Enfermedad , Emodina/administración & dosificación , Emodina/farmacología , Técnicas de Silenciamiento del Gen , Homeostasis/efectos de los fármacos , Metalotioneína/genética , Metales/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Quercetina/administración & dosificación , Quercetina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Oxidative stress is commonly observed in both idiopathic and genetic cases of Parkinson's disease (PD). It plays an important role in the degeneration of dopaminergic neurons, and it has been associated with altered telomere length (TL). There is currently no cure for PD, and extracts of antioxidative plant, such as Mucuna pruriens and Withania somnifera, are commonly used in Ayurveda to treat patients with PD. In this study, we evaluated 2 enzymatic markers of oxidative stress, glutathione (GSH) system and superoxide dismutase (SOD), and TL in a Drosophila melanogaster model for PD [phosphatase and tensin homolog-induced putative kinase 1 (PINK1)B9]. This evaluation was also performed after treatment with the phytoextracts. PINK1B9 mutants showed a decrease in GSH amount and SOD activity and unexpected longer telomeres compared with wild-type flies. M. pruriens treatment seemed to have a beneficial effect on the oxidative stress conditions. On the other hand, W. somnifera treatment did not show any improvements in the studied oxidative stress mechanisms and even seemed to favor the selection of flies with longer telomeres. In summary, our study suggests the importance of testing antioxidant phytoextracts in a PINK1B9 model to identify beneficial effects for PD.-Baroli, B., Loi, E., Solari, P., Kasture, A., Moi, L., Muroni, P., Kasture, S., Setzu, M. D., Liscia, A., Zavattari, P. Evaluation of oxidative stress mechanisms and the effects of phytotherapic extracts on Parkinson's disease Drosophila PINK1B9 model.
Asunto(s)
Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Extractos Vegetales/farmacología , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster/metabolismo , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , Proteínas Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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 HEK293â¯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'.
Asunto(s)
Encefalopatías Metabólicas Innatas/tratamiento farmacológico , Creatina/deficiencia , Discapacidad Intelectual Ligada al Cromosoma X/tratamiento farmacológico , Proteínas del Tejido Nervioso/efectos de los fármacos , Fenilbutiratos/farmacología , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficiencia , Deficiencias en la Proteostasis/tratamiento farmacológico , Animales , Encefalopatías Metabólicas Innatas/genética , Calnexina/metabolismo , Membrana Celular/metabolismo , Creatina/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Células HEK293 , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Cultivo Primario de Células , Deficiencias en la Proteostasis/genética , RatasRESUMEN
The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. Here we introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-R607A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R599A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. We explored the possible physiological role of somatodendritic SERT by comparing flies harboring wild type SERT and dSERT-R599A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. We conclude that, in CSD neurons, (i) somatodendritic SERT supports ethanol attraction, (ii) axonal SERT specifies ethanol aversion, (iii) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Asunto(s)
Axones/fisiología , Dendritas/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Olfato/fisiología , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Línea Celular , Depresores del Sistema Nervioso Central/farmacología , Dendritas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Etanol/farmacología , Humanos , Interneuronas/efectos de los fármacos , Mutación Puntual/genética , Cultivo Primario de Células , Pliegue de Proteína , Núcleos del Rafe/citología , Núcleos del Rafe/metabolismo , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Olfato/genéticaRESUMEN
Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Fenómenos Electrofisiológicos/efectos de los fármacos , Locomoción/efectos de los fármacos , Fitoquímicos/farmacología , Extractos Vegetales/farmacología , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/fisiopatología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Mutación , Fitoquímicos/química , Extractos Vegetales/química , Proteinopatías TDP-43/tratamiento farmacológicoRESUMEN
The brain of Drosophila melanogaster is comprised of some 100,000 neurons, 127 and 80 of which are dopaminergic and serotonergic, respectively. Their activity regulates behavioral functions equivalent to those in mammals, e.g., motor activity, reward and aversion, memory formation, feeding, sexual appetite, etc. Mammalian dopaminergic and serotonergic neurons are known to be heterogeneous. They differ in their projections and in their gene expression profile. A sophisticated genetic tool box is available, which allows for targeting virtually any gene with amazing precision in Drosophila melanogaster. Similarly, Drosophila genes can be replaced by their human orthologs including disease-associated alleles. Finally, genetic manipulation can be restricted to single fly neurons. This has allowed for addressing the role of individual neurons in circuits, which determine attraction and aversion, sleep and arousal, odor preference, etc. Flies harboring mutated human orthologs provide models which can be interrogated to understand the effect of the mutant protein on cell fate and neuronal connectivity. These models are also useful for proof-of-concept studies to examine the corrective action of therapeutic strategies. Finally, experiments in Drosophila can be readily scaled up to an extent, which allows for drug screening with reasonably high throughput.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Drosophila melanogaster/fisiología , Neuronas Serotoninérgicas/fisiología , Transmisión Sináptica , Animales , Neuronas Dopaminérgicas/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuronas Serotoninérgicas/metabolismoRESUMEN
It has been suggested that the dentate gyrus, particularly its new neurons generated via adult neurogenesis, is involved in memory acquisition and recall. Here, we trained rats in two types of Morris water maze tasks that are differentially associated with these two memory processes, and examined whether new neurons are differently affected by the two tasks performed during the second week of neuronal birth. Our results indicate that the task involving more opportunities to acquire new information better supports the survival of new neurons. Further, we assessed whether the two tasks differentially induce the expression of an immediate early gene, Zif268, which is known to be induced by neuronal activation. While the two tasks differentially induce Zif268 expression in the dentate gyrus, the proportions of new neurons activated were similar between the two tasks. Thus, we conclude that while the two tasks differentially activate the dentate gyrus, the task involving more opportunities for memory acquisition during the second week of the birth of new neurons better promotes the survival of the new neurons.
Asunto(s)
Conducta Animal/fisiología , Giro Dentado/citología , Memoria , Neuronas/citología , Animales , Supervivencia Celular , Giro Dentado/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Femenino , Regulación de la Expresión Génica , Aprendizaje por Laberinto/fisiología , RatasRESUMEN
Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum when heterologously expressed in HEK293 cells. In three of these mutants, i.e. hDAT-V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit was remedied with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin-µ such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or pifithrin-µ treatment supported hDAT delivery to the presynaptic terminals of dopaminergic neurons and restored sleep to normal length in DAT-deficient (fumin) Drosophila lines expressing hDAT-V158F or hDAT-G327R. In contrast, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, indicating a toxic action not remedied by pharmacochaperoning. Our observations identified those mutations most likely amenable to pharmacological rescue in the affected children. In addition, our findings also highlight the challenges of translating insights from pharmacochaperoning in cell culture to the clinical situation. Because of the evolutionary conservation in dopaminergic neurotransmission between Drosophila and people, pharmacochaperoning of DAT in D. melanogaster may allow us to bridge that gap.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Drosophila melanogaster/efectos de los fármacos , Ibogaína/análogos & derivados , Mutación , Trastornos Parkinsonianos/tratamiento farmacológico , Sulfonamidas/farmacología , Animales , Animales Modificados Genéticamente , Conducta Animal/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Humanos , Ibogaína/farmacología , Masculino , Trastornos Parkinsonianos/genética , Transmisión SinápticaRESUMEN
The key role of monoamine transporters is to take up neurotransmitters from the synaptic cleft and rapidly terminate neurotransmission. Monoamine transporters begin their journey by folding in the endoplasmic reticulum. Upon achieving their natively-folded state, the oligomerized transporters engage the coat protein complex II machinery and exit the endoplasmic reticulum compartment in a concentrative fashion. The transporters are subsequently sorted in the endoplasmic reticulum-Golgi intermediate complex and the Golgi apparatus, prior to reaching their pivotal site of action at the plasma membrane. Stringent quality-control mechanisms ensure that only the correctly-folded protein cargo departs the endoplasmic reticulum. Genetic point mutations in the coding sequences of monoamine transporters can trigger severe physiologic deficiencies by inducing folding defects. Protein misfolding precludes the delivery of functional monoamine transporters to the cell surface. Chemical- and/or pharmacological-chaperone molecules, which facilitate folding, have proven effective in restoring the activity of several misfolded pathological variants of monoamine transporters.
Asunto(s)
Proteínas de Transporte de Neurotransmisores/química , Proteínas de Transporte de Neurotransmisores/metabolismo , Pliegue de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Retículo Endoplásmico/metabolismo , Humanos , Terapia Molecular DirigidaRESUMEN
Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to µm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants.
Asunto(s)
Chaperonas Moleculares/química , Naftoles/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Sustitución de Aminoácidos , Células HEK293 , Humanos , Transporte Iónico , Membrana Dobles de Lípidos/química , Chaperonas Moleculares/farmacología , Mutación Missense , Naftoles/farmacología , Conformación Proteica , Pliegue de Proteína , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sodio/química , Sodio/metabolismoRESUMEN
Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-µ. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-µ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-µ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Ibogaína/análogos & derivados , Pliegue de Proteína/efectos de los fármacos , Sueño/genética , Sulfonamidas/farmacología , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Humanos , Ibogaína/administración & dosificación , Ibogaína/farmacología , Simulación de Dinámica Molecular , Mutación , Fenotipo , Sulfonamidas/administración & dosificaciónRESUMEN
The human dopamine transporter (hDAT) belongs to the solute carrier 6 (SLC6) gene family. Point mutations in hDAT (SLC6A3) have been linked to a syndrome of dopamine transporter deficiency or infantile dystonia/parkinsonism. The mutations impair DAT folding, causing retention of variant DATs in the endoplasmic reticulum and subsequently impair transport activity. The folding trajectory of DAT itself is not understood, though many insights have been gained from studies of folding-deficient mutants of the closely related serotonin transporter (SERT); i.e. their functional rescue by pharmacochaperoning with (nor)ibogaine or heat-shock protein inhibitors. We recently provided a proof-of-principle that folding-deficits in DAT are amenable to rescue in vitro and in vivo. As a model we used the Drosophila melanogaster DAT mutant dDAT-G108Q, which phenocopies the fumin/sleepless DAT-knockout. Treatment with noribogaine and/or HSP70 inhibitor pifithrin-µ restored folding of, and dopamine transport by, dDAT-G108Q, its axonal delivery and normal sleep time in mutant flies. The possibility of functional rescue of misfolded DATs in living flies by pharmacochaperoning grants new therapeutic prospects in the remedy of folding diseases, not only in hDAT, but also in other SLC6 transporters, in particular mutants of the creatine transporter-1, which give rise to X-linked mental retardation.
RESUMEN
In the traditional system of medicine in Sardinia, Rubia peregrina (RP) is reported as an aphrodisiac herb. Since aphrodisiacs may also have dopaminergic activity, and there can be a reciprocal relationship between dopaminergic and serotonergic functions in the central nervous system, the aim here was to study the effect of the ethanolic extract of the aerial parts of R. peregrina on monoamine-mediated behaviour. The RP (100 and 200 mg kg(-1) intraperitoneally) significantly inhibited haloperidol (1 mg kg(-1) i.p.) induced catalepsy in mice (p < 0.01, dopamine-mediated response) lithium sulphate induced head twitches in rats (serotonin-mediated response) and was without effect on clonidine-induced hypothermia in rats (noradrenaline-mediated response). The study concludes that R. peregrina improves the dopaminergic function, diminishes the serotonin-mediated function and is devoid of any effect on the noradrenergic function. The study suggests that we should carry out further experiments to investigate the psychopharmacological profile of R. peregrina.
Asunto(s)
Dopaminérgicos/farmacología , Componentes Aéreos de las Plantas/química , Extractos Vegetales/farmacología , Rubia/química , Serotoninérgicos/farmacología , Adrenérgicos/farmacología , Animales , Catalepsia/tratamiento farmacológico , Discinesias/tratamiento farmacológico , Haloperidol , Compuestos de Litio , Masculino , Ratones , Extractos Vegetales/química , Psicofarmacología , Ratas , SulfatosRESUMEN
Neuroleptic-induced tardive dyskinesia (TD) is a motor disorder of the orofacial region resulting from chronic neuroleptic treatment. The agents improving dopaminergic transmission improve TD. Mucuna pruriens seed contains levodopa and amino acids. The effect of methanolic extract of M. pruriens seeds (MEMP) was studied on haloperidol-induced TD, alongside the changes in lipid peroxidation, reduced glutathione, superoxide dismutase (SOD) and catalase levels. The effect of MEMP was also evaluated in terms of the generation of hydroxyl and 1,1-diphenyl,2-picrylhydrazyl (DPPH) radical. MEMP (100 and 200 mg kg⻹) inhibited haloperidol-induced vacuous chewing movements, orofacial bursts and biochemical changes. MEMP also inhibited hydroxyl radical generation and DPPH. The results of the present study suggest that MEMP by virtue of its free radical scavenging activity prevents neuroleptic-induced TD.