[Show abstract][Hide abstract] ABSTRACT: 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 3 mg/ml) in the drinking water during 5 weeks. We found that 3 mg/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, 0.5 mg/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.
Full-text · Article · Apr 2016 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Nowadays it is assumed that besides its roles in neuronal processing, dopamine (DA) is also involved in the regulation of cerebral blood flow. However, studies on the hemodynamic actions of DA have been mainly focused on the cerebral cortex, but the possibility that vessels in deeper brain structures receive dopaminergic axons and the origin of these axons have not been investigated. Bearing in mind the evidence of changes in the blood flow of basal ganglia in Parkinson's disease (PD), and the pivotal role of the dopaminergic mesostriatal pathway in the pathophysiology of this disease, here we studied whether striatal vessels receive inputs from midbrain dopaminergic neurons. The injection of an anterograde neuronal tracer in combination with immunohistochemistry for dopaminergic, vascular and astroglial markers, and dopaminergic lesions, revealed that midbrain dopaminergic axons are in close apposition to striatal vessels and perivascular astrocytes. These axons form dense perivascular plexuses restricted to striatal regions in rats and monkeys. Interestingly, they are intensely immunoreactive for tyrosine hydroxylase (TH) phosphorylated at Ser19 and Ser40 residues. The presence of phosphorylated TH in vessel terminals indicates they are probably the main source of basal TH activity in the striatum, and that after activation of midbrain dopaminergic neurons, DA release onto vessels precedes that onto neurons. Furthermore, the relative weight of this "vascular component" within the mesostriatal pathway suggests that it plays a relevant role in the pathophysiology of PD.
Full-text · Article · Aug 2014 · Frontiers in Neuroanatomy
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Dec 2010 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: The term vulnerability was first associated with the midbrain dopaminergic neurons 85 years ago, before they were identified as monoaminergic neurons, when Foix and Nicolesco (1925) reported the loss of neuromelanin containing neurons in the midbrain of patients with post-encephalitic Parkinson's disease (PD). A few years later, Hassler (1938) showed that degeneration is more intense in the ventral tier of the substantia nigra compacta than in its dorsal tier and the ventral tegmental area (VTA), outlining the concept of differential vulnerability of midbrain dopaminergic (DA-) neurons. Nowadays, we know that other neuronal groups degenerate in PD, but the massive loss of nigral DA-cells is its pathological hallmark, having a pivotal position in the pathophysiology of the disease as it is responsible for the motor symptoms. Data from humans as well as cellular and animal models indicate that DA-cell degeneration is a complex process, probably precipitated by the convergence of different risk factors, mediated by oxidative stress, and involving pathogenic factors arising within the DA-neuron (intrinsic factors), and from its environment and distant interconnected brain regions (extrinsic factors). In light of current data, intrinsic factors seem to be preferentially involved in the first steps of the degenerative process, and extrinsic factors in its progression. A controversial issue is the relative weight of the impairment of common cell functions, such as energy metabolism and proteostasis, and specific dopaminergic functions, such as pacemaking activity and DA handling, in the pathogenesis of DA-cell degeneration. Here we will review the current knowledge about the relevance of these factors at the beginning and during the progression of PD, and in the differential vulnerability of midbrain DA-cells.
Full-text · Article · Oct 2010 · Frontiers in Neuroanatomy
[Show abstract][Hide abstract] ABSTRACT: The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δβγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.
Full-text · Article · Oct 2010 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.
Full-text · Article · Sep 2009 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: The degeneration of nigral dopaminergic (DA-) neurons is the histopathologic hallmark of Parkinson's disease (PD), but not all nigral DA-cells show the same susceptibility to degeneration. This starts in DA-cells in the ventrolateral and caudal regions of the susbtantia nigra (SN) and progresses to DA-cells in the dorsomedial and rostral regions of the SN and the ventral tegmental area, where many neurons remain intact until the final stages of the disease. This fact indicates a relationship between the topographic distribution of midbrain DA-cells and their differential vulnerability, and the possibility that this differential vulnerability is associated with phenotypic differences between different subpopulations of nigral DA-cells. Studies carried out during the last two decades have contributed to establishing the existence of different compartments of nigral DA-cells according to their neurochemical profile, and a possible relationship between the expression of some factors and the relative vulnerability or resistance of DA-cell subpopulations to degeneration. These aspects are reviewed and discussed here.
No preview · Article · Jan 2009 · Journal of neural transmission. Supplementum
[Show abstract][Hide abstract] ABSTRACT: Several studies report that the striatal dopamine (DA) uptake declines with age, but the underlying mechanisms are still unclear. The use of molecular, biochemical and morphological techniques, and antibodies which detect the glycosylated (80 kDa) and non-glycosylated (50 kDa) DA transporter (DAT) forms in the rat mesostriatal system, reveals that DAT is pre- and post-translationally damaged during aging. In middle age (18 months), the glycosylated DAT form decreases in the plasma membrane of striatal terminals, and the non-glycosylated form is accumulated in the endoplasmic reticulum-Golgi complex. Thereafter, in aged rats (24 months), DAT synthesis is also affected as the decrease in both DATmRNA and total DAT protein levels suggests. However, the evidence of a decrease in both DAT expression in the endosomal (vesicle-enriched) compartment and the phosphorylated DAT fraction from middle age, as well as its compartmental redistribution towards the terminal plasma membrane, with an increase in the membrane DAT/total DAT ratio in striatal synapotosomes, in aged rats, indicate that DA-cells activate compensatory mechanisms directed at maintaining DAT function during normal aging.
No preview · Article · Nov 2007 · Neurobiology of aging
[Show abstract][Hide abstract] ABSTRACT: Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them.
Full-text · Article · Sep 2007 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: The vesicular monoamine transporter type 2 (VMAT2) is a transmembrane glycoprotein responsible for the vesicular monoamine uptake in the brain. This function declines in the dopaminergic mesostriatal system during normal aging, but the mechanisms responsible for this deficit are unknown. We investigated possible age-related changes in the expression and subcellular distribution of VMAT2 in the rat mesostriatal system. VMAT2 is constitutively expressed as glycosylated (75 kDa), partially glycosylated (55 kDa) and native (45 kDa) forms, they are all present in both synaptosomal compartments (synaptosomal membrane and synaptic vesicle-enriched fractions) of the striatal terminals in young rats. In aged rats, no changes were found in midbrain VMAT2mRNA and VMAT2 total protein levels in whole striatal extracts. However, its subcellular distribution and glycosylation pattern were severely modified. The three VMAT2 forms virtually disappeared from the synaptic vesicle-enriched fraction, while the 55 kDa form was accumulated in the soluble compartment. These changes may be responsible for the loss of VMAT2 activity during aging and may contribute to the high susceptibility of aged midbrain dopaminergic cells to degeneration.
No preview · Article · Jul 2007 · Neurobiology of aging
[Show abstract][Hide abstract] ABSTRACT: The impairment of the mesostriatal dopaminergic system has been considered responsible for motor and affective disturbances associated with aging and a risk factor for Parkinson's disease. However, the basic mechanisms underlying this phenomenon are still unknown. Here we used biochemical, molecular and morphological techniques directed at detecting flaws in the dopamine synthesis route and signs of dopaminergic degeneration in the rat mesostriatal system during normal aging. We found two different age-related processes. One is characterized by a dopa decarboxylase decrease, and involves both the nigrostriatal and mesolimbic compartments, and is responsible for a moderate dopamine loss in the dorsal striatum, where other parameters of dopamine synthesis are not affected. The other is characterized by axonal degeneration with aggregation of phosphorylated forms of tyrosine hydroxylase (TH) and amyloid precursor protein in degenerate terminals, and alpha-synuclein in their original somata. This process is restricted to mesolimbic regions and is responsible for the decline of TH activity and l-dopa levels and the greater decrease in dopamine levels in this compartment. These findings suggest that both the nigrostriatal and the mesolimbic systems are vulnerable to aging, but in contrast to what occurs in Parkinson's disease, the mesolimbic system is more vulnerable to aging than the nigrostriatal one.
No preview · Article · Apr 2007 · Experimental Neurology
[Show abstract][Hide abstract] ABSTRACT: In the present study we have analyzed the effect of tetrahydrobiopterin (BH4) essential cofactor for tyrosine hydroxylase and nitric oxide synthase, on the 3,4-dihydroxyphenylalanine (L-DOPA) release from in vitro incubated striatal tissue. dl-6-methyl-5,6,7,8 tetrahydropterine (6-MPH4)-stimulated L-DOPA release in a concentration-dependent manner in the range from 25 to 100 microM. At these concentrations 6-MPH4 did not have any effect on dopamine release. Presence of Nomega-Nitro-L-arginine methyl ester (L-NAME, 200 microM), a nitric oxide synthase inhibitor, but not of alpha-methyl-rho-tyrosine (alpha-MPT, 100 microM), a tyrosine hydroxylase inhibitor, blocked L-DOPA release induced by 6-MPH4 (200 microM). Also, the addition to the incubation medium of melatonin (MEL, 300 microM), which is a scavenger of NO and other free radicals, blocked the L-DOPA release induced by 6-MPH4 (200 microM) but this effect did not occur with the addition of the peroxynitrite scavenger uric acid (UA, 300 microM). Sodium nitroprusside (SNP, 100 muM), a NO generator and l-DOPA releaser as previously reported, potentiated the L-DOPA releasing effect of 6-MPH4 (200 microM) which was also blocked by melatonin. In summary 6-MPH4 stimulates L-DOPA release from striatal fragments incubated in vitro by a mechanism which involves NO or other free radicals derived from NO but not peroxynitrite.
No preview · Article · Aug 2006 · European Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide synthase (NOS) and interleukin-6 (IL-6) are constitutively expressed in hypothalamic cells. However, phenotypic and functional aspects of these cells remain unknown. We have studied the expression pattern of these two molecules in hypothalamic cells expressing corticotropin-releasing factor (CRF) and arginin-vasopressin (AVP), two major regulatory peptides in the hypothalamus-pituitary system, using immunofluorescence, intracerebroventricular injection of colchicine, and the study in parallel of the labeling pattern of axons in the median eminence. Within AVP cells, we distinguished two different populations: large, intensely stained AVP cells coexpressing IL-6; and large, intensely stained AVP cells coexpressing IL-6 and NOS. Within the CRF cells, we distinguished three different populations: large, intensely stained CRF cells immunonegative for AVP, NOS, and IL-6; large cells weakly stained for CRF and AVP, immunopositive for NOS and immunonegative for IL-6; and small cells intensely stained for CRF and AVP and immunonegative for IL-6 and NOS. In addition, we also found AVP cells containing IL-6 in the suprachiasmatic nucleus. These results suggest that neuronal NOS and IL-6 may be involved in different modulatory processes in hypophysiotropic and non-hypophysiotropic cells.
Full-text · Article · May 2006 · Journal of Histochemistry and Cytochemistry
[Show abstract][Hide abstract] ABSTRACT: Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRalpha1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro-gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro-gold, and after colchicine injection in the lateral ventricle, they become GDNF-immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6-OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.
Full-text · Article · May 2005 · European Journal of Neuroscience