RESUMEN
The dopamine transporter (DAT) is a membrane glycoprotein in dopaminergic neurons, which modulates extracellular and intracellular dopamine levels. DAT is regulated by different presynaptic proteins, including dopamine D2 (D2R) and D3 (D3R) receptors. While D2R signalling enhances DAT activity, some data suggest that D3R has a biphasic effect. However, despite the extensive therapeutic use of D2R/D3R agonists in neuropsychiatric disorders, this phenomenon has been little studied. In order to shed light on this issue, DAT activity, expression and posttranslational modifications were studied in mice and DAT-D3R-transfected HEK cells. Consistent with previous reports, acute treatment with D2R/D3R agonists promoted DAT recruitment to the plasma membrane and an increase in DA uptake. However, when the treatment was prolonged, DA uptake and total striatal DAT protein declined below basal levels. These effects were inhibited in mice by genetic and pharmacological inactivation of D3R, but not D2R, indicating that they are D3R-dependent. No changes were detected in mesostriatal tyrosine hydroxylase (TH) protein expression and midbrain TH and DAT mRNAs, suggesting that the dopaminergic system is intact and DAT is posttranslationally regulated. The use of immunoprecipitation and cell surface biotinylation revealed that DAT is phosphorylated at serine residues, ubiquitinated and released into late endosomes through a PKCß-dependent mechanism. In sum, the results indicate that long-term D3R activation promotes DAT down-regulation, an effect that may underlie neuroprotective and antidepressant actions described for some D2R/D3R agonists.
Asunto(s)
Agonistas de Dopamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteína Quinasa C/metabolismo , Proteolisis/efectos de los fármacos , Receptores de Dopamina D3/metabolismo , Ubiquitinación/fisiología , Animales , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pramipexol/farmacología , Receptores de Dopamina D3/agonistas , Ubiquitinación/efectos de los fármacosRESUMEN
The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.
Asunto(s)
Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Análisis de Varianza , Animales , Membrana Celular/metabolismo , Cuerpo Estriado/citología , Dopamina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Ligadura , Masculino , Ratas , Ratas Sprague-Dawley , Transducción Genética , Tritio/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization.
Asunto(s)
Cuerpo Estriado/enzimología , Trastornos Parkinsonianos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Dopamina D1/metabolismo , Animales , Benzazepinas/farmacología , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Agonistas de Dopamina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Trastornos Parkinsonianos/enzimología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
The dopamine (DA) transporter (DAT), a membrane glycoprotein expressed in dopaminergic neurons, clears DA from extracellular space and is regulated by diverse presynaptic proteins like protein kinases, α-synuclein, D2 and D3 autoreceptors. DAT dysfunction is implicated in Parkinson's disease and depression, which are therapeutically treated by dopaminergic D2/D3 receptor (D2/D3R) agonists. It is, then, important to improve our understanding of interactions between D3R and DAT. We show that prolonged administration of pramipexole (0.1mg/kg/day, 6 to 21 days), a preferential D3R agonist, leads to a decrease in DA uptake in mouse striatum that reflects a reduction in DAT affinity for DA in the absence of any change in DAT density or subcellular distribution. The effect of pramipexole was absent in mice with genetically-deleted D3R (D3R(-/-)), yet unaffected in mice genetically deprived of D2R (D2R(-/-)). Pramipexole treatment induced a physical interaction between D3R and DAT, as assessed by co-immunoprecipitation and in situ proximity ligation assay. Furthermore, it promoted the formation of DAT dimers and DAT association with both D2R and α-synuclein, effects that were abolished in D3R(-/-) mice, yet unaffected in D2R(-/-) mice, indicating dependence upon D3R. Collectively, these data suggest that prolonged treatment with dopaminergic D3 agonists provokes a reduction in DA reuptake by dopaminergic neurons related to a hitherto-unsuspected modification of the DAT interactome. These observations provide novel insights into the long-term antiparkinson, antidepressant and additional clinical actions of pramipexole and other D3R agonists.
Asunto(s)
Autorreceptores/metabolismo , Benzotiazoles/farmacología , Cuerpo Estriado/efectos de los fármacos , Agonistas de Dopamina/farmacología , Dopamina/metabolismo , Receptores de Dopamina D3/metabolismo , Animales , Antidepresivos/farmacología , Antiparkinsonianos/farmacología , Cuerpo Estriado/metabolismo , Dimerización , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Pramipexol , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/genética , alfa-Sinucleína/metabolismoRESUMEN
Aberrant membrane localization of dopamine D(1) receptor (D1R) is associated with L-DOPA-induced dyskinesia (LID), a major complication of L-DOPA treatment in Parkinson's disease (PD). Since the proteasome plays a central role in modulating neuronal response through regulation of neurotransmitter receptor intraneuronal fate, we hypothesized that the ubiquitine-proteasome proteolytic pathway could be impaired in LID. Those LIDs are actually associated with a striatum-specific decrease in proteasome catalytic activity and accumulation of polyubiquitinated proteins in experimental rodent and monkey parkinsonism. We then demonstrated that such decreased proteasome catalytic activity (1) results from D1R activation and (2) feed-back the D1R abnormal trafficking, i.e., its exaggerated cell surface abundance. We further showed that the genetic invalidation of the E3 ubiquitin-protein ligase parkin PD gene leads to exaggerated abnormal involuntary movements compared with wild-type mice. We thus established in an unprecedented series of experimental models that impairment of the ubiquitine-proteasome system at specific nodes (E3 ligase parkin, polyubiquitination, proteasome catalytic activity) leads to the same phenomenon, i.e., aberrant behavioral response to dopamine replacement therapy in PD, highlighting the intimate interplay between dopamine receptor and proteasome activity in a nondegenerative context.
Asunto(s)
Discinesia Inducida por Medicamentos/metabolismo , Levodopa/toxicidad , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Receptores de Dopamina D1/agonistas , Animales , Modelos Animales de Enfermedad , Agonistas de Dopamina/toxicidad , Discinesia Inducida por Medicamentos/fisiopatología , Femenino , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Trastornos Parkinsonianos/enzimología , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/fisiologíaRESUMEN
Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.
Asunto(s)
Globo Pálido/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Núcleo Accumbens/metabolismo , Glándula Pineal/metabolismo , Multimerización de Proteína/fisiología , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Masculino , Proteínas del Tejido Nervioso/genética , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genéticaRESUMEN
BACKGROUND: The anterior lobe of the insular cortex (aINS) is a cortical region that has reciprocal connections with limbic centers such as the anterior cingulate cortex, prefrontal cortex, amygdala and nucleus accumbens (NAc). In fact, the aINS has been involved in the integration of autonomic information for emotional and motivational functions. The compulsive consumption of drugs or high-fat foods induces alterations at both behavioural and brain levels. Brain reward circuits are altered in response to continued intake, in particular the dopaminergic projections from the ventral tegmental area (VTA) to the NAc. The aINS has multiple connections with the components of this system. In recent years, efforts have been made to better understand the fundamental role of the aINS in addiction, making it one of the key centres of interest for research into new treatments for addiction. OBJECTIVES: The present work focuses on studying 1.- whether the human aINS expresses orexigenic peptides such as neuropeptide Y (NPY), a peptide known to induce hyperphagia, and which has been implicated in the onset and development of obesity, 2.- the long-term effect of an obesogenic diet on NPY expression in the aINS and NAc of C57BL/6 mice. METHODS: A total of 17 female C57BL/6 J mice were used in this study. Female mice were fed ad libitum with water and, either a standard diet (SD) or a high-fat diet (HFD) to induce obesity. There were seven female mice on the SD and ten on the HFD. The duration of the experiment was 180 days. We also studied 3 human adult brains (1 male and 2 females, mean age 55.7 ± 5.2 years). The morphological study was performed using immunohistochemistry and double immunofluorescence techniques to study the neurochemical profile of NPY neurons of the aINS and NAc of humans and mice. RESULTS: Our morphological analysis demonstrates for the first time the basal expression of NPY in different layers of the human cortex (II, III, IV, V/VI), in a pattern similar to previous studies in other species. Furthermore, we observed an increase in the number of NPY-positive cells and their intracytoplasmic signal in the aINS and NAc of the obese mice subjected to a long-term obesogenic diet. CONCLUSIONS: To our knowledge, this is the first study to show the distribution and expression of NPY in the human INS and how its expression is altered after prolonged treatment with an obesogenic diet in obese mice. Our findings may contribute to the understanding of the pathophysiological mechanisms underlying obesity in regions related to the reward system and associated with uncontrolled intake of high-fat foods, thus facilitating the identification of novel therapeutic targets.
Asunto(s)
Neuropéptido Y , Núcleo Accumbens , Humanos , Ratones , Masculino , Femenino , Animales , Persona de Mediana Edad , Núcleo Accumbens/metabolismo , Ratones Obesos , Corteza Insular , Ratones Endogámicos C57BL , Obesidad/metabolismo , Dieta Alta en Grasa/efectos adversosRESUMEN
BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that affects primarily the dopaminergic (DAergic) neurons of the mesostriatal system, among other nuclei of the brain. Although it is considered an idiopathic disease, oxidative stress is believed to be involved in DAergic neuron death and therefore plays an important role in the onset and development of the disease. RAD9B is a paralog of the RAD9 checkpoint, sharing some similar functions related to DNA damage resistance and apoptosis, as well as the ability to form 9-1-1 heterotrimers with RAD1 and HUS1. METHODS: In addition to immunohistochemistry, immunofluorescence and Western-blot analysis, we implemented Quantitative RT-PCR and in situ hybridization techniques. RESULTS: We demonstrated RAD9B expression in rat and human mesencephalic DAergic cells using specific markers. Additionally, we observed significant overexpression of RAD9B mRNA (p<0.01) and protein (p<0.01) in the midbrain 48 h after inducing damage with 150 µg of 6-hydroxydopamine (6-OHDA) injected in a rat model of PD. Regarding protein expression, the increased levels were observed in neurons of the mesostriatal system and returned to normal 5 days post-injury. CONCLUSIONS: This response to a neurotoxin, known to produce oxidative stress specifically on DAergic neurons indicates the potential importance of RAD9B in this highly vulnerable population to cell death. In this model, RAD9B function appears to provide neuroprotection, as the induced lesion resulted in only mild degeneration. This observation highlights the potential of RAD9B checkpoint protein as a valuable target for future therapeutic interventions aimed at promoting neuroprotection.
Asunto(s)
Enfermedad de Parkinson , Animales , Humanos , Ratas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopamina/uso terapéutico , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Estrés Oxidativo , Oxidopamina/toxicidad , Oxidopamina/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismoRESUMEN
The suprachiasmatic nucleus (SCN) is the main region for the regulation of circadian rhythms. Although the SCN contains a heterogeneous neurochemical phenotype with a wide variety of neuropeptides, a key role has been suggested for the vasoactive intestinal neuropeptide (VIP) as a modulator circadian, reproductive, and seasonal rhythms. VIP is a 28-amino acid polypeptide hormone that belongs to the secretin-glucagon peptide superfamily and shares 68 % homology with the pituitary adenylate cyclase-activating polypeptide (PACAP). VIP acts as an endogenous appetite inhibitor in the central nervous system, where it participates in the control of appetite and energy homeostasis. In recent years, significant efforts have been made to better understand the role of VIP in the regulation of appetite/satiety and energy balance. This study aimed to elucidate the long-term effect of an obesogenic diet on the distribution and expression pattern of VIP in the SCN and nucleus accumbens (NAc) of C57BL/6 mice. A total of 15 female C57BL/6J mice were used in this study. Female mice were fed ad libitum with water and, either a standard diet (SD) or a high-fat diet (HFD) to induce obesity. There were 7 female mice on the SD and 8 on the HFD. The duration of the experiment was 365 days. The morphological study was performed using immunohistochemistry and double immunofluorescence techniques to study the neurochemical profile of VIP neurons of the SCN of C57BL/6 mice. Our data show that HFD-fed mice gained weight and showed reduced VIP expression in neurons of the SCN and also in fibres located in the NAc. Moreover, we observed a loss of neuropeptide Y (NPY) expression in fibres surrounding the SCN. Our findings on VIP may contribute to the understanding of the pathophysiological mechanisms underlying obesity in regions associated with uncontrolled intake of high-fat foods and the reward system, thus facilitating the identification of novel therapeutic targets.
Asunto(s)
Dieta Alta en Grasa , Péptido Intestinal Vasoactivo , Femenino , Animales , Ratones , Ratones Endogámicos C57BL , Dieta Alta en Grasa/efectos adversos , Ratones Obesos , Núcleo SupraquiasmáticoRESUMEN
Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinson's disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson's disease-like and l-DOPA-treated Parkinson's disease-like conditions in experimental (rat and monkey) and human Parkinson's disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinson's disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson's disease.
Asunto(s)
Encéfalo/metabolismo , Trastornos Parkinsonianos/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Anciano , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/ultraestructura , Colina O-Acetiltransferasa/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Dendritas/metabolismo , Dendritas/ultraestructura , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopaminérgicos/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/deficiencia , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Humanos , Levodopa/farmacología , Macaca fascicularis , Masculino , Haz Prosencefálico Medial/efectos de los fármacos , Haz Prosencefálico Medial/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Inmunoelectrónica/métodos , Persona de Mediana Edad , Factores de Crecimiento Nervioso/metabolismo , Compuestos Orgánicos/metabolismo , Oxidopamina/efectos adversos , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/patología , Cambios Post Mortem , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Espectrometría de Masas en Tándem , alfa-Metiltirosina/farmacologíaRESUMEN
The dopamine transporter (DAT) is a transmembrane glycoprotein responsible for dopamine (DA) uptake, which has been shown to be involved in DA-cell degeneration in Parkinson's disease (PD). At the same time, some studies suggest that DAT may be regulated in response to dopaminergic injury. We have investigated the mechanisms underlying DAT regulation after different degrees of dopaminergic lesion. DAT is persistently down-regulated in surviving midbrain DA-neurons after substantial (62%) loss of striatal DA-terminals, and transiently after slight (11%) loss of DA-terminals in rats. Transient DAT down-regulation consisted of a decrease of glycosylated (mature) DAT in the plasma membrane with accumulation of non-glycosylated (immature) DAT in the endoplasmic reticulum-Golgi (ERG) compartment, and recovery of the normal expression pattern 5 days after lesion. DAT redistribution to the ERG was also observed in HEK cells expressing rat DAT exposed to MPP(+), but not after exposure to DAT-unrelated neurotoxins. In contrast to other midbrain DA-cells, those in the ventrolateral region of the substantia nigra do not regulate DAT and degenerate shortly after slight DA-lesion. These data suggest that DAT down-regulation is a post-translational event induced by DA-analogue toxins, consisting of a stop in its glycosylation and trafficking to the plasma membrane. Its persistence after substantial DA-lesion may act as a compensatory mechanism helping maintain striatal DA levels. The fact that neurons which do not regulate DAT die shortly after lesion suggests a relationship between DAT down-regulation and neuroprotection.
Asunto(s)
Adrenérgicos/toxicidad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Regulación de la Expresión Génica , Oxidopamina/toxicidad , Adrenérgicos/administración & dosificación , Animales , Western Blotting , Regulación hacia Abajo , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Inmunohistoquímica , Hibridación in Situ , Inyecciones Intraventriculares , Oxidopamina/administración & dosificación , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The current basal ganglia model considers the internal division of the globus pallidus and the substantia nigra pars reticulata as the sole sources of basal ganglia output to the thalamus. However, following the delivery of retrograde tracers into the ventral anterior/ventral lateral thalamic nuclei, a moderate number of labeled neurons were found within the subthalamic nucleus (STN) in control cases, MPTP-treated monkeys and animals with levodopa-induced dyskinesias. Furthermore, dual tracing experiments showed that subthalamo-thalamic and subthalamo-pallidal projections arise from different subpopulations of STN efferent neurons. Moreover, upregulated expression of the mRNA coding the vesicular glutamate transporter 2 (vGlut2) was found in retrogradely-labeled STN neurons in MPTP-treated monkeys. By contrast, there is a reduction in vGlut2 mRNA expression in subthalamo-thalamic neurons in dyskinetic monkeys. In conclusion, our findings support the presence of a direct projection from the STN to the ventral thalamus that appears to be functionally modulated by dopaminergic activity.
Asunto(s)
Macaca fascicularis/fisiología , Enfermedad de Parkinson Secundaria/fisiopatología , Núcleo Subtalámico/fisiología , Núcleos Talámicos Ventrales/fisiología , Animales , Masculino , Microscopía Confocal , Vías Nerviosas/fisiología , Trazadores del Tracto Neuronal , Neuronas/metabolismo , Enfermedad de Parkinson Secundaria/inducido químicamente , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Growing evidence shows that autophagy is deficient in neurodegenerative and psychiatric diseases, and that its induction may have beneficial effects in these conditions. However, as autophagy shares signaling pathways with cell death and interferes with protein synthesis, prolonged use of autophagy inducers available nowadays is considered unwise. The search for novel autophagy inducers indicates that DRD2 (dopamine receptor 2)-DRD3 ligands may also activate autophagy, though critical aspects of the action mechanisms and effects of dopamine ligands on autophagy are still unknown. In order to shed light on this issue, DRD2- and DRD3-overexpressing cells and drd2 KO, drd3 KO and wild-type mice were treated with the DRD2-DRD3 agonist pramipexole. The results revealed that pramipexole induces autophagy through MTOR inhibition and a DRD3-dependent but DRD2-independent mechanism. DRD3 activated AMPK followed by inhibitory phosphorylation of RPTOR, MTORC1 and RPS6KB1 inhibition and ULK1 activation. Interestingly, despite RPS6KB1 inhibition, the activity of RPS6 was maintained through activation of the MAPK1/3-RPS6KA pathway, and the activity of MTORC1 kinase target EIF4EBP1 along with protein synthesis and cell viability, were also preserved. This pattern of autophagy through MTORC1 inhibition without suppression of protein synthesis, contrasts with that of direct allosteric and catalytic MTOR inhibitors and opens up new opportunities for G protein-coupled receptor ligands as autophagy inducers in the treatment of neurodegenerative and psychiatric diseases. ABBREVIATIONS: AKT/Protein kinase B: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; BECN1: beclin 1; EGFP: enhanced green fluorescent protein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GPCR; G protein-coupled receptor; GFP: green fluorescent protein; HEK: human embryonic kidney; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MDA: malonildialdehyde; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPX: pramipexole; RPTOR/raptor: regulatory associated protein of MTOR, complex 1; RPS6: ribosomal protein S6; RPS6KA/p90S6K: ribosomal protein S6 kinase A; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.
Asunto(s)
Autofagia , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Biosíntesis de Proteínas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagia/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacos , Pramipexol/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proto-Oncogenes Mas , Proteína S6 Ribosómica/metabolismo , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.
Asunto(s)
Globo Pálido/fisiopatología , Interneuronas/fisiología , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/fisiopatología , Animales , Calbindina 2 , Recuento de Células , Colina O-Acetiltransferasa/metabolismo , Densitometría , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Óxido Nítrico Sintasa de Tipo I/metabolismo , Oxidopamina , Parvalbúminas/metabolismo , Virus de la Rabia , Ratas , Ratas Wistar , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.
Asunto(s)
Globo Pálido/metabolismo , Ácido Glutámico/metabolismo , Trastornos Parkinsonianos/metabolismo , Tálamo/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Dopamina/deficiencia , Regulación hacia Abajo/fisiología , Vías Eferentes/metabolismo , Vías Eferentes/fisiopatología , Núcleo Entopeduncular/metabolismo , Núcleo Entopeduncular/fisiopatología , Regulación Enzimológica de la Expresión Génica/genética , Globo Pálido/fisiopatología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Masculino , Trastornos Parkinsonianos/fisiopatología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Sustancia Negra/metabolismo , Sustancia Negra/fisiopatología , Transmisión Sináptica/fisiología , Tálamo/fisiopatología , Regulación hacia Arriba/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/genéticaRESUMEN
The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.
Asunto(s)
Corteza Entorrinal/citología , Neuronas/citología , Terminales Presinápticos/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Animales , Biotina/análogos & derivados , Biotina/metabolismo , Mapeo Encefálico , Calbindina 2 , Dextranos/metabolismo , Femenino , Imagenología Tridimensional/métodos , Hibridación in Situ/métodos , Microscopía Confocal/métodos , Vías Nerviosas/fisiología , Parvalbúminas/metabolismo , Ratas , Ratas Wistar , Estilbamidinas/metabolismoRESUMEN
The present study is focused on the analysis of the vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) used by thalamic neurons giving rise to the thalamostriatal system. Instead of studying the distribution of VGLUT proteins at the level of thalamostriatal terminals, this report is focused on identifying the expression of the VGLUT mRNAs within the parent cell bodies of thalamic neurons innervating the striatum. For this purpose, we have combined dual in situ hybridization to detect both VGLUT1 and VGLUT2 mRNAs together with retrograde tracing with cholera toxin. Our results show that VGLUT2 is the only vesicular glutamate transporter expressed in thalamostriatal-projecting neurons located in the midline and intralaminar nuclei, whereas all neurons from the ventral thalamic nuclei innervating the striatum express both VGLUTs, at least at the mRNA level. Indeed, the mRNAs encoding for VGLUT1 and VGLUT2 displayed a sharp complementary subcellular distribution within neurons from the ventral thalamic nuclei giving rise to thalamostriatal projections. The differential distribution of VGLUT mRNAs lead us to conclude that the thalamostriatal pathway is a dual system, composed by a preponderant projection arising from the midline and intralaminar nuclei using VGLUT2 as the glutamate transporter, together with another important source of striatal afferents arising from neurons in the ventral thalamic relay nuclei containing both kinds of vesicular glutamate transporters.
Asunto(s)
Cuerpo Estriado/metabolismo , Ácido Glutámico/metabolismo , Tálamo/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/genética , Animales , Mapeo Encefálico , Toxina del Cólera/metabolismo , Cuerpo Estriado/citología , Inmunohistoquímica , Hibridación Fluorescente in Situ , Núcleos Talámicos Intralaminares/citología , Núcleos Talámicos Intralaminares/metabolismo , Masculino , Núcleos Talámicos de la Línea Media/citología , Núcleos Talámicos de la Línea Media/metabolismo , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Coloración y Etiquetado , Transmisión Sináptica/fisiología , Tálamo/citologíaRESUMEN
The caudal intralaminar nuclei are a major source of glutamatergic afferents to the basal ganglia. Experiments in the 6-hydroxydopamine rat model have shown that the parafascicular nucleus is overactive and its lesion alleviates basal ganglia neurochemical abnormalities associated with dopamine depletion. Accordingly, removal of this excitatory innervation of the basal ganglia could have a beneficial value in the parkinsonian state. To test this hypothesis, unilateral kainate-induced chemical ablation of the centromedian thalamic nucleus (CM) has been performed in MPTP-treated monkeys. Successful lesions restricted to the CM boundaries (n = 2) without spreading over other neighboring thalamic nuclei showed an initial, short-lasting, and mild change in the parkinsonian motor scale but no effect against levodopa-induced dyskinesias. The lack of significant and persistent motor improvement leads us to conclude that unilateral selective lesion of the CM alone cannot be considered as a suitable surgical approach for the treatment of PD or levodopa-induced dyskinesias. The role of the caudal intralaminar nuclei in the pathophysiology of movement disorders of basal ganglia origin remains to be clarified.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Núcleos Talámicos Intralaminares/efectos de los fármacos , Ácido Kaínico/toxicidad , Trastornos Parkinsonianos/fisiopatología , Animales , Discinesia Inducida por Medicamentos/diagnóstico , Lateralidad Funcional/efectos de los fármacos , Núcleos Talámicos Intralaminares/patología , Levodopa/efectos adversos , Levodopa/uso terapéutico , Macaca fascicularis , Masculino , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Técnicas EstereotáxicasRESUMEN
Although currently available retrograde tracers are useful tools for identifying striatal projection neurons, transported tracers often remained restricted within the neuronal somata and the thickest, main dendrites. Indeed, thin dendrites located far away from the cell soma as well as post-synaptic elements such as dendritic spines cannot be labeled unless performing intracellular injections. In this regard, the subsequent use of anterograde tracers for the labeling of striatal afferents often failed to unequivocally elucidate whether a given afferent makes true contacts with striatal projections neurons. Here we show that such a technical constraint can now be circumvented by retrograde tracing using rabies virus (RV). Immunofluorescence detection with a monoclonal antibody directed against the viral phosphoprotein resulted in a consistent Golgi-like labeling of striatal projection neurons, allowing clear visualization of small-size elements such as thin dendrites as well as dendritic spines. The combination of this retrograde tracing together with dual anterograde tracing of cortical and thalamic afferents has proven to be a useful tool for ascertaining striatal microcircuits. Indeed, by taking advantage of the trans-synaptic spread of RV, different subpopulations of local-circuit neurons modulating striatal efferent neurons can also be identified. At the striatal level, structures displaying labeling were visualized under the confocal laser-scanning microscope at high resolution. Once acquired, confocal stacks of images were firstly deconvoluted and then processed through 3D-volume rendering in order to unequivocally identify true contacts between pre-synaptic elements (axon terminals from cortical or thalamic sources) and post-synaptic elements (projection neurons and/or interneurons labeled with RV).
Asunto(s)
Mapeo Encefálico/métodos , Cuerpo Estriado/citología , Red Nerviosa/citología , Neuronas/citología , Virus de la Rabia/inmunología , Coloración y Etiquetado/métodos , Animales , Anticuerpos Monoclonales/inmunología , Transporte Axonal/fisiología , Cuerpo Estriado/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Técnica del Anticuerpo Fluorescente/métodos , Interneuronas/citología , Interneuronas/metabolismo , Masculino , Microscopía Confocal , Red Nerviosa/metabolismo , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Neuroanatomía/métodos , Neuronas/metabolismo , Neuronas/virología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Virus de la Rabia/metabolismo , Ratas , Ratas Wistar , Transmisión Sináptica/fisiología , Proteínas Virales/inmunologíaRESUMEN
Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.