RESUMEN
Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.
Asunto(s)
Microscopía por Crioelectrón , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas de Transporte Vesicular de Monoaminas , Animales , Microscopía por Crioelectrón/métodos , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Proteínas de Transporte Vesicular de Monoaminas/química , Ingeniería de Proteínas/métodos , Ovinos , Humanos , Simulación del Acoplamiento Molecular , Células HEK293RESUMEN
BACKGROUND: High-frequency repeated transcranial magnetic stimulation (rTMS) stimulating the primary motor cortex (M1) is an alternative, adjunctive therapy for improving the motor symptoms of Parkinson's disease (PD). However, whether the high frequency of rTMS positively correlates to the improvement of motor symptoms of PD is still undecided. By controlling for other parameters, a disease animal model may be useful to compare the neuroprotective effects of different high frequencies of rTMS. OBJECTIVE: The current exploratory study was designed to compare the protective effects of four common high frequencies of rTMS (5, 10, 15, and 20 Hz) and iTBS (a special form of high-frequency rTMS) and explore the optimal high-frequency rTMS on an animal PD model. METHODS: Following high frequencies of rTMS application (twice a week for 5 weeks) in a MPTP/probenecid-induced chronic PD model, the effects of the five protocols on motor behavior as well as dopaminergic neuron degeneration levels were identified. The underlying molecular mechanisms were further explored. RESULTS: We found that all the high frequencies of rTMS had protective effects on the motor functions of PD models to varying degrees. Among them, the 10, 15, and 20 Hz rTMS interventions induced comparable preservation of motor function through the protection of nigrostriatal dopamine neurons. The enhancement of brain-derived neurotrophic factor (BDNF), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT-2) and the suppression of TNF-α and IL-1ß in the nigrostriatum were involved in the process. The efficacy of iTBS was inferior to that of the above three protocols. The effect of 5 Hz rTMS protocol was weakest. CONCLUSIONS: Combined with the results of the present study and the possible side effects induced by rTMS, we concluded that 10 Hz might be the optimal stimulation frequency for preserving the motor functions of PD models using rTMS treatment.
Asunto(s)
Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Trastornos Parkinsonianos , Probenecid , Estimulación Magnética Transcraneal , Animales , Estimulación Magnética Transcraneal/métodos , Ratones , Masculino , Probenecid/farmacología , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/terapia , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Motora/metabolismo , Corteza Motora/fisiopatología , Neuronas Dopaminérgicas/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Interleucina-1beta/metabolismo , Sustancia Negra/metabolismo , Cuerpo Estriado/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Intoxicación por MPTP/terapia , Intoxicación por MPTP/prevención & control , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/fisiopatología , Actividad Motora/fisiología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacologíaRESUMEN
INTRODUCTION: Tardive dyskinesia (TD) and Huntington's disease (HD)-associated chorea are persistent and disabling hyperkinetic disorders that can be treated with vesicular monoamine transporter type 2 (VMAT2) inhibitors, including the recently approved once-daily (QD) formulation of deutetrabenazine (DTBZ ER). While its efficacy and safety profile have not been directly investigated, currently available data confirms bioequivalence and similar bioavailability to the twice-daily formulation (DTBZ BID). AREAS COVERED: The authors briefly review the pivotal trials establishing efficacy of DTBZ for TD and HD-associated chorea, the pharmacokinetic data for bioequivalence between QD and BID dosing of DTBZ, as well as dose proportionality evidence, titration recommendations, and safety profile for DTBZ ER. EXPERT OPINION: Long-term data show that DTBZ is efficacious and well tolerated for the treatment of TD and HD-associated chorea. DTBZ ER likely demonstrates therapeutic equivalence with no new safety signals. Due to the lack of comparative clinical trial data, no evidence-based recommendation about choice of VMAT2 inhibitor or switching between VMAT2 inhibitors can be made about best practice. Ultimately, QD dosing may offer the chance of improved medication adherence, an important consideration in patients with complex treatment regimens and/or patients with cognitive decline.
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Preparaciones de Acción Retardada , Enfermedad de Huntington , Discinesia Tardía , Tetrabenazina , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/complicaciones , Discinesia Tardía/tratamiento farmacológico , Tetrabenazina/análogos & derivados , Tetrabenazina/uso terapéutico , Tetrabenazina/administración & dosificación , Tetrabenazina/farmacocinética , Tetrabenazina/efectos adversos , Corea/tratamiento farmacológico , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Inhibidores de Captación Adrenérgica/administración & dosificación , Inhibidores de Captación Adrenérgica/uso terapéutico , Inhibidores de Captación Adrenérgica/farmacocinética , Inhibidores de Captación Adrenérgica/efectos adversos , ComprimidosRESUMEN
Neuromuscular junctions are innervated by motor and sympathetic nerves. The sympathetic modulation of motor innervation shows functional decline during aging, but the cellular and molecular mechanism of this change is not fully known. This study aimed to evaluate the effect of aging on sympathetic nerves and synaptic proteins at mouse neuromuscular junctions. Sympathetic nerves, presynaptic, and postsynaptic proteins of sympathetic nerves at neuromuscular junctions were visualized using immunohistochemistry, and aging-related changes were compared between adult-, aged-, and nicotinamide mononucleotide (NMN) administered aged mice. Sympathetic nerves were detected by anti-tyrosine hydroxylase antibody, and presynaptic protein vesicular monoamine transporter 2 colocalized with the sympathetic nerves. These two signals surrounded motor nerve terminals and acetylcholine receptor clusters. Postsynaptic neurotransmitter receptor ß2-adrenergic receptors colocalized with motor nerve terminals and resided in reduced density at extrasynaptic sarcolemma. The signal intensity of the sympathetic nerve marker did not show a significant difference at neuromuscular junctions between 8.5-month-old adult mice and 25-month-old aged mice. However, the signal intensity of vesicular monoamine transporter 2 and ß2-adrenergic receptors showed age-related decline at neuromuscular junctions. Interestingly, both age-related declines reverted to the adult level after 1 month of oral administration of NMN by drinking water. In contrast, NMN administration did not alter the expression level of sympathetic marker tyrosine hydroxylase at neuromuscular junctions. The results suggest a functional decline of sympathetic nerves at aged neuromuscular junctions due to decreases in presynaptic and postsynaptic proteins, which can be reverted to the adult level by NMN administration.
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Envejecimiento , Unión Neuromuscular , Mononucleótido de Nicotinamida , Animales , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Envejecimiento/metabolismo , Envejecimiento/efectos de los fármacos , Ratones , Mononucleótido de Nicotinamida/farmacología , Mononucleótido de Nicotinamida/administración & dosificación , Masculino , Terminales Presinápticos/metabolismo , Terminales Presinápticos/efectos de los fármacos , Ratones Endogámicos C57BL , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
Several genetic pathogenic variants increase the risk of Parkinson's disease (PD) with pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene being among the most common. A joint pattern analysis based on multi-set canonical correlation analysis (MCCA) was utilized to extract PD and LRRK2 pathogenic variant-specific spatial patterns in relation to healthy controls (HCs) from multi-tracer Positron Emission Tomography (PET) data. Spatial patterns were extracted for individual subject cohorts, as well as for pooled subject cohorts, to explore whether complementary spatial patterns of dopaminergic denervation are different in the asymptomatic and symptomatic stages of PD. The MCCA results are also compared to the traditional univariate analysis, which serves as a reference. We identified PD-induced spatial distribution alterations common to DAT and VMAT2 in both asymptomatic LRRK2 pathogenic variant carriers and PD subjects. The inclusion of HCs in the analysis demonstrated that the dominant common PD-induced pattern is related to an overall dopaminergic terminal density denervation, followed by asymmetry and rostro-caudal gradient with deficits in the less affected side still being the best marker of disease progression. The analysis was able to capture a trend towards PD-related patterns in the LRRK2 pathogenic variant carrier cohort with increasing age in line with the known increased risk of this patient cohort to develop PD as they age. The advantage of this method thus resides in its ability to identify not only regional differences in tracer binding between groups, but also common disease-related alterations in the spatial distribution patterns of tracer binding, thus potentially capturing more complex aspects of disease induced alterations.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson , Tomografía de Emisión de Positrones , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/diagnóstico por imagen , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Tomografía de Emisión de Positrones/métodos , Persona de Mediana Edad , Femenino , Masculino , Anciano , Adulto , Heterocigoto , Encéfalo/diagnóstico por imagen , Proteínas de Transporte Vesicular de Monoaminas/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genéticaRESUMEN
BACKGROUND: Reserpine (RES), a Vesicular Monoamine Transporter 2 (VMAT2) inhibitor agent, has been used in preclinical research for many years to create animal models for depression and to test experimental antidepressant strategies. Nevertheless, evidence of the potential use and validity of RES as a chronic pharmacological model for depression is lacking, and there are no comprehensive studies of the behavioral effects in conjunction with molecular outcomes. METHODS: Experiment 1. Following baseline behavior testing sensitive to depression-like phenotype and locomotion (Phase 1), 27 Sprague-Dawley (SD) rats received i.p. either vehicle solution (0.0 mg/kg), low (0.2 mg/kg) or high (0.8 mg/kg) RES dose for 20 days using a pre-determined schedule and reassessed for behavioral phenotypes (Phase 2). After 10 days washout period, and a final behavioral assessment (Phase 3), the brains were collected 16 days after the last injection for mRNA-expression assessment. Experiment 2. In a similar timetable as in Experiment 1 but without the behavioral testing, 12 SD rats underwent repetitive dopamine D2/3 receptor PET scanning with [18F]DMFP following each Phase. The binding potential (BPND) of [18F]DMFP was quantified by kinetic analysis as a marker of striatal D2/3R availability. Weight and welfare were monitored throughout the study. RESULTS: Significant, dose-dependent weight loss and behavioral deficits including both motor (hypo-locomotion) and non-motor behavior (anhedonia, mild anxiety and reduced exploration) were found for both the low and high dose groups with significant decrease in D2R mRNA expression in the accumbal region for the low RES group after Phase 3. Both RES treated groups showed substantial increase in [18F]DMFP BPND (in line with dopamine depletion) during Phase 2 and 3 compared to baseline and Controls. CONCLUSIONS: The longitudinal design of the study demonstrated that chronic RES administration induced striatal dopamine depletion that persisted even after the wash-out period. However, the behavior phenotype observed were transient. The data suggest that RES administration can induce a rodent model for depression with mild face validity.
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Depresión , Modelos Animales de Enfermedad , Tomografía de Emisión de Positrones , Ratas Sprague-Dawley , Reserpina , Animales , Reserpina/farmacología , Masculino , Ratas , Depresión/inducido químicamente , Depresión/metabolismo , Conducta Animal/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Relación Dosis-Respuesta a Droga , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Actividad Motora/efectos de los fármacosRESUMEN
Amphetamines (Amph) are psychostimulants broadly used as physical and cognitive enhancers. However, the long-term effects of prenatal exposure to Amph have been poorly investigated. Here, we show that continuous exposure to Amph during early development induces long-lasting changes in histone methylation at the C. elegans tyrosine hydroxylase (TH) homolog cat-2 and the vesicular monoamine transporter (VMAT) homologue cat-1 genes. These Amph-induced histone modifications are correlated with enhanced expression and function of CAT-2/TH and higher levels of dopamine, but decreased expression of CAT-1/VMAT in adult animals. Moreover, while adult animals pre-exposed to Amph do not show obvious behavioral defects, when challenged with Amph they exhibit Amph hypersensitivity, which is associated with a rapid increase in cat-2/TH mRNA. Because C. elegans has helped reveal neuronal and epigenetic mechanisms that are shared among animals as diverse as roundworms and humans, and because of the evolutionary conservation of the dopaminergic response to psychostimulants, data collected in this study could help us to identify the mechanisms through which Amph induces long-lasting physiological and behavioral changes in mammals.
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Anfetamina , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Desarrollo Embrionario , Tirosina 3-Monooxigenasa , Proteínas de Transporte Vesicular de Monoaminas , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Anfetamina/farmacología , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Dopamina/metabolismo , Epigénesis Genética/efectos de los fármacosRESUMEN
Vesicular monoamine transporter (VMAT)-2 has a crucial role in the neurotransmission of biogenic amines. Recently, Dalton et al., Pidathala et al., Wu et al., and Wang et al. individually reported cryo-electron microscopy (EM) structures of human VMAT2, offering opportunities for developing improved therapeutics and deep insights into the functioning of this protein.
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Microscopía por Crioelectrón , Desarrollo de Medicamentos , Proteínas de Transporte Vesicular de Monoaminas , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Humanos , AnimalesRESUMEN
Background: Tardive Dyskinesia (TD) is a neurological disorder characterized by involuntary movements, often caused by dopamine receptor antagonists. Vesicular Monoamine Transporter 2 (VMAT2) inhibitors, such as valbenazine and deutetrabenazine, have emerged as promising therapies for TD and several clinical trials have shown their efficacy. This study aims to compare the efficacy and safety profile of VMAT2 inhibitors, focusing on a recent trial conducted in the Asian population. Methods: We reviewed the PubMed, Cochrane Library, Embase database, and clinicaltrials.gov between January 2017 and October 2023, using the keywords "tardive dyskinesia" AND ("valbenazine" [all fields] OR " deutetrabenazine " [all fields]) AND "clinical trial". The reviewed articles were studied for efficacy and side effects. Results: An initial search yielded 230 articles, of which 104 were duplicates. Following the title and abstract screening, 25 additional articles were excluded. A full-text review resulted in the exclusion of 96 more articles. Ultimately, four double-blind clinical trials met the inclusion criteria. The deutetrabenazine studies demonstrated significant improvements in Abnormal Involuntary Movement Scale (AIMS) scores compared to placebo, with no difference in adverse events. The valbenazine studies showed favorable results in reducing TD symptoms and were well-tolerated. Discussion: The studies reviewed in this analysis underscore the potential of deutetrabenazine and valbenazine as valuable treatment options for TD in diverse populations. Both medications demonstrated significant improvements in AIMS scores, suggesting their effectiveness in managing TD symptoms. Additionally, they exhibited favorable safety profiles, with low rates of serious adverse events and no significant increase in QT prolongation, parkinsonism, suicidal ideation, or mortality. Conclusion: The studies reviewed highlight the promising efficacy and tolerability of deutetrabenazine and valbenazine as treatments for Tardive Dyskinesia, providing new hope for individuals affected by this challenging condition.
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Discinesia Tardía , Tetrabenazina , Valina , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Discinesia Tardía/tratamiento farmacológico , Discinesia Tardía/inducido químicamente , Tetrabenazina/efectos adversos , Tetrabenazina/análogos & derivados , Tetrabenazina/uso terapéutico , Valina/análogos & derivados , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Neuronas Dopaminérgicas , Sistema Nervioso Entérico , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Neuronas Dopaminérgicas/metabolismo , Ratones , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/citología , Ratones Transgénicos , Tirosina 3-Monooxigenasa/metabolismo , Dopamina/metabolismo , Masculino , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
BACKGROUND: As a biomarker targeting vesicular monoamine transporter 2 (VMAT2), 18F-9-fluoropropyldihydrotetrabenazine (18F-FP-DTBZ) positron emission tomography (PET) is highly accurate in diagnosing Parkinson's disease (PD) and assessing its severity. However, evidence is insufficient in patients with progressive supranuclear palsy (PSP). OBJECTIVE: We evaluated the striatal and extrastriatal monoaminergic disruption of PSP and differences in patterns between patients with PSP, PD, and healthy controls (HCs) using 18F-FP-DTBZ PET, as well as its correlations with the clinical characteristics of PSP. METHODS: We recruited 58 patients with PSP, 23 age- and duration-matched patients with PD, as well as 17 HCs. Patients were scanned using 18F-FP-DTBZ PET/computed tomography, and images were spatially normalized and analyzed based on the volume of interest. RESULTS: VMAT2 binding differed significantly in the striatum and substantia nigra among the groups (P < 0.001). A more severe disruption in the caudate was noted in the PSP group (P < 0.001) than in the PD group. However, no differences were found in the nucleus accumbens, hippocampus, amygdala, or raphe between the PD and PSP groups. Within the PSP group, striatal VMAT2 binding was significantly associated with the fall/postural stability subscore of the PSP Rating Scale, especially in the putamen. Furthermore, VMAT2 binding was correlated with Mini-Mental State Examination or Montreal Cognitive Assessment in the hippocampus. CONCLUSIONS: Caudate disruptions showed prominent differences among the groups. VAMT2 binding in the striatum and hippocampus reflects the severity of fall/postural stability and cognition, respectively. © 2024 International Parkinson and Movement Disorder Society.
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Cuerpo Estriado , Enfermedad de Parkinson , Parálisis Supranuclear Progresiva , Proteínas de Transporte Vesicular de Monoaminas , Humanos , Parálisis Supranuclear Progresiva/diagnóstico por imagen , Parálisis Supranuclear Progresiva/metabolismo , Masculino , Femenino , Anciano , Persona de Mediana Edad , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Cuerpo Estriado/metabolismo , Cuerpo Estriado/diagnóstico por imagen , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Tetrabenazina/análogos & derivados , Sustancia Negra/diagnóstico por imagen , Sustancia Negra/metabolismo , Sustancia Negra/patología , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodosRESUMEN
Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.
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Monoaminas Biogénicas , Interacciones Farmacológicas , Proteínas de Transporte Vesicular de Monoaminas , Humanos , 1-Metil-4-fenilpiridinio/química , 1-Metil-4-fenilpiridinio/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Anfetamina/química , Anfetamina/farmacología , Anfetamina/metabolismo , Sitios de Unión , Monoaminas Biogénicas/química , Monoaminas Biogénicas/metabolismo , Microscopía por Crioelectrón , Dopamina/química , Dopamina/metabolismo , Modelos Moleculares , Norepinefrina/química , Norepinefrina/metabolismo , Unión Proteica , Protones , Reserpina/farmacología , Reserpina/química , Reserpina/metabolismo , Serotonina/química , Serotonina/metabolismo , Especificidad por Sustrato , Proteínas de Transporte Vesicular de Monoaminas/química , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/ultraestructuraRESUMEN
OBJECTIVES: Current treatments for Parkinson's disease using pharmacological approaches alleviate motor symptoms but do not prevent neuronal loss or dysregulation of dopamine neurotransmission. In this article, we have explored the molecular mechanisms underlying the neuroprotective effect of the antioxidant N-acetylcysteine (NAC) on the damaged dopamine system. METHODS: SH-SY5Y cells were differentiated towards a dopaminergic phenotype and exposed to 6-hydroxydopamine (6-OHDA) to establish an in vitro model of Parkinson's disease. We examined the potential of NAC to restore the pathological effects of 6-OHDA on cell survival, dopamine synthesis as well as on key proteins regulating dopamine metabolism. Specifically, we evaluated gene- and protein expression of tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and α-synuclein, by using qPCR and Western blot techniques. Moreover, we quantified the effect of NAC on total dopamine levels using a dopamine ELISA assay. RESULTS: Our results indicate that NAC has a neuroprotective role in SH-SY5Y cells exposed to 6-OHDA by maintaining cell proliferation and decreasing apoptosis. Additionally, we demonstrated that NAC treatment increases dopamine release and protects SH-SY5Y cells against 6-OHDA dysregulations on the proteins TH, VMAT2, and α-synuclein. CONCLUSIONS: Our findings contribute to the validation of compounds capable to restore dopamine homeostasis and shed light on the metabolic pathways that could be targeted to normalize dopamine turnover. Furthermore, our results highlight the effectiveness of the antioxidant NAC in the prevention of dopaminergic neurodegeneration in the present model. ABBREVIATIONS: DAT, dopamine transporter; 6-OHDA, 6-hydroxydopamine; NAC, N-acetylcysteine; PARP, poly (ADP-ribose) polymerase; RA; retinoic acid; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; VMAT2, vesicle monoamine transporter 2.
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Acetilcisteína , Dopamina , Oxidopamina , Tirosina 3-Monooxigenasa , Proteínas de Transporte Vesicular de Monoaminas , alfa-Sinucleína , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Humanos , Oxidopamina/toxicidad , alfa-Sinucleína/metabolismo , Dopamina/metabolismo , Acetilcisteína/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Línea Celular Tumoral , Fármacos Neuroprotectores/farmacología , Supervivencia Celular/efectos de los fármacosRESUMEN
The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.
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Enfermedad de Huntington , Tetrabenazina , Humanos , Tetrabenazina/metabolismo , Tetrabenazina/farmacología , Proteínas de Transporte Vesicular de Monoaminas/química , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Protones , Microscopía por CrioelectrónRESUMEN
ß-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupê·containing amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupê·containing amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.
Asunto(s)
Aminoácidos Diaminos , Aminoácidos , Aminoácidos/farmacología , Proteínas de Transporte Vesicular de Monoaminas , Aminoácidos Diaminos/toxicidad , Tirosina , Neurotoxinas/metabolismoRESUMEN
RATIONALE: Dopamine antagonists induce dopamine receptor supersensitivity. This may manifest in late-appearing movement disorders (tardive dyskinesia (TD). VMAT-2 inhibitors reduce dopaminergic transmission but have limited activity at postsynaptic receptors and so may have antipsychotic activity with lower risk of tardive dyskinesia. METHODS: We conducted a systematic database search from inception to September 2022 for articles describing the use of VMAT-2 inhibitors in psychosis. Inclusion criteria were as follows: Population: adults diagnosed with psychosis or schizophrenia; Intervention: treatment with tetrabenazine, deutetrabenazine or valbenazine; Comparison: comparison with placebo or/and antipsychotic drug; Outcomes: with efficacy outcomes (e.g. Brief Psychiatric Rating Scale (BPRS) change or clinician assessment) and adverse effects ratings (e.g. rating scale or clinician assessment or dropouts); and Studies: in randomised controlled trials and non-randomised studies. RESULTS: We identified 4892 records relating to VMAT-2 inhibitor use of which 5 (173 participants) met our a priori meta-analysis inclusion criteria. VMAT-2 inhibitors were more effective than placebo for the outcome 'slight improvement' (risk ratio (RR) = 1.77 (95% CI 1.03, 3.04)) but not for 'moderate improvement' (RR 2.81 (95% CI 0.27, 29.17). VMAT-2 inhibitors were as effective as active comparators on both measures for-'slight improvement' (RR 1.05 (95% CI 0.6, 1.81)) and 'moderate improvement' (RR 1.11 (95% CI 0.51, 2.42). Antipsychotic efficacy was also suggested by a narrative review of 37 studies excluded from the meta-analysis. CONCLUSIONS: VMAT-2 inhibitors may have antipsychotic activity and may offer promise for treatment of psychosis with the potential for a reduced risk of TD.
Asunto(s)
Antipsicóticos , Trastornos Psicóticos , Esquizofrenia , Proteínas de Transporte Vesicular de Monoaminas , Adulto , Humanos , Antipsicóticos/efectos adversos , Trastornos Psicóticos/tratamiento farmacológico , Esquizofrenia/tratamiento farmacológico , Discinesia Tardía/tratamiento farmacológico , Tetrabenazina/uso terapéutico , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidoresRESUMEN
Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.
Asunto(s)
Tetrabenazina , Proteínas de Transporte Vesicular de Monoaminas , Humanos , Reserpina , Serotonina/metabolismo , Vesículas Sinápticas/metabolismo , Tetrabenazina/farmacología , Tetrabenazina/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1-3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression4-6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Transporte Vesicular de Monoaminas , Humanos , Sitios de Unión , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Ketanserina/química , Ketanserina/metabolismo , Ketanserina/farmacología , Reserpina/química , Reserpina/metabolismo , Reserpina/farmacología , Serotonina/química , Serotonina/metabolismo , Especificidad por Sustrato , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacología , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Monoaminas/química , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/ultraestructuraRESUMEN
BACKGROUND: Prior studies suggest that norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) mediate meta-iodobenzylguanidine (MIBG) uptake and retention in neuroblastoma tumors. We evaluated the relationship between NET and VMAT2 tumor expression and clinical response to 131 I-MIBG therapy in patients with neuroblastoma. METHODS: Immunohistochemistry (IHC) was used to evaluate NET and VMAT2 protein expression levels on archival tumor samples (obtained at diagnosis or relapse) from patients with relapsed or refractory neuroblastoma treated with 131 I-MIBG. A composite protein expression H-score was determined by multiplying a semi-quantitative intensity value (0-3+) by the percentage of tumor cells expressing the protein. RESULTS: Tumor samples and clinical data were available for 106 patients, of whom 28.3% had partial response (PR) or higher. NET H-score was not significantly associated with response (≥PR), though the percentage of tumor cells expressing NET was lower among responders (median 80% for ≥PR vs. 90% for Asunto(s)
3-Yodobencilguanidina
, Neuroblastoma
, Humanos
, 3-Yodobencilguanidina/uso terapéutico
, Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo
, Proteínas de Transporte Vesicular de Monoaminas/metabolismo
, Radiofármacos
, Proteína Proto-Oncogénica N-Myc
, Recurrencia Local de Neoplasia/tratamiento farmacológico
, Neuroblastoma/tratamiento farmacológico
, Enfermedad Crónica