Ignacio Cruz-Muros

Striatal expression of GDNF and differential vulnerability of midbrain dopaminergic cells.

Glial cell line‐derived neurotrophic factor (GDNF) is a member of the transforming growth factor‐β 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 Parkinsons 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 (GFRα1 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 Parkinsons 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.

Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinsons 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.

Vulnerability of mesostriatal dopaminergic neurons in Parkinson's disease.

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 Parkinsons 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.

Aging effects on the dopamine transporter expression and compensatory mechanisms

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.

Aging of the rat mesostriatal system: Differences between the nigrostriatal and the mesolimbic compartments

The impairment of the mesostriatal dopaminergic system has been considered responsible for motor and affective disturbances associated with aging and a risk factor for Parkinsons 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 Parkinsons disease, the mesolimbic system is more vulnerable to aging than the nigrostriatal one.

Interleukin-6 and Nitric Oxide Synthase Expression in the Vasopressin and Corticotrophin-releasing Factor Systems of the Rat Hypothalamus

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.

Deglycosylation and subcellular redistribution of VMAT2 in the mesostriatal system during normal aging

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.

The dopamine transporter is differentially regulated after dopaminergic lesion.

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 Parkinsons 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.

Prolonged treatment with pramipexole promotes physical interaction of striatal dopamine D3 autoreceptors with dopamine transporters to reduce dopamine uptake

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 Parkinsons 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.

Effect of intracerebroventricular injection of lipopolysaccharide on the tuberoinfundibular dopaminergic system of the rat

Numerous studies indicate that monoaminergic systems are sensitive to both peripheral and central inflammatory stimuli, and in particular dopaminergic neurons in the nigrostriatal system degenerate after local injection of lipopolysaccharide (LPS). However, data about the response of other dopaminergic groups to local inflammation are very sparse. In this study, we have examined the effect of i.c.v. injection of LPS on the tuberoinfundibular dopaminergic (TIDA) system by using biochemical and morphological parameters. Our results show that 6 h after i.c.v. injection of LPS, in parallel to a transient and intense immunoreaction to interleukin-1beta in arcuate microglial cells, there is a decrease in tyrosine hydroxylase (TH) activity in the median eminence and in the number of TH- and TH mRNA-positive cells in the arcuate nucleus, and at 12 h, an increase of prolactin levels in serum. Posterior changes were found in the TH mRNA labeling pattern, mostly in the ventrolateral region of the arcuate nucleus, but they were not accompanied by any changes in TH activity and immunoreactivity and TH-cell count. This suggests that the TIDA system is functionally susceptible to local inflammation, but the effects are transient and do not induce neurodegeneration.