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Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease
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.
... Cultures were maintained in a humidified incubator set at 37 • C and 5% CO 2 . Stable cells expressing rat DAT were obtained as described in Afonso-Oramas et al. [35]. ...
... Different commercial anti-DAT antibodies against C-and N-terminal fragments of human and rat DAT have been tested in our laboratory using brain samples of different mammalians and cells transfected with wild type and mutated DAT forms [23,35,38,39]. In order to confirm their specificity and sensitivity, and which of them provides better efficiency in the different techniques further tests have been performed (see supplementary Fig.1). ...
... For biotinylation experiments, synaptosomes were obtained following the impermeant biotinylation procedure [23,35]. Striatal samples were immediately homogenized in 20 vol of ice-cold sucrose bicarbonate solution (SBS, 320 mM sucrose (#S0385; Sigma) in 5 mM sodium bicarbonate (#S5761; Sigma), pH 7.4) with 12 up and down strokes in a Teflon-glass homogenizer. ...
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 PKCβ-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.
... However, hints of heterogeneity have emerged with regard to the levels of expression and activity of even these defining molecular markers. In the case of DAT, a consensus has emerged from various studies and in several species, that it is more highly expressed in SNc DA neurons compared to VTA DA neurons [58][59][60][61][62][63]. Within the VTA, at least in rodents, the dorsolateral regions appear to express reasonable levels of DAT; ventromedially however, Dat was found to be very low or absent in some VTA neurons [64]. ...
... Similarly, in one study on human midbrain, DAT was detectable only in a little more than half of VTA DA neurons [59]. Some studies have also detected variations in levels of glycosylated DAT, considered the active form of the transporter [58,65]. In rodents, glycosylated DAT is highest in the SNc as well as the dorsolateral VTA. ...
... Within the human SNc, more ventral tier DA neurons contain glycosylated DAT [59], whereas dorsal tier SNc or VTA neurons had less glycosylated DAT. Consistent with these findings, levels of glycosylated DAT were shown to be higher in the terminals of nigrostriatal DA neurons than in mesolimbic DA neurons [58]. In the case of VMAT2/ SLC18A2, expression was detected in most DA neurons [59,66]. ...
Since their discovery, midbrain dopamine (DA) neurons have been researched extensively, in part because of their diverse functions and involvement in various neuropsychiatric disorders. Over the last few decades, reports have emerged that midbrain DA neurons were not a homogeneous group, but that DA neurons located in distinct anatomical locations within the midbrain had distinctive properties in terms of physiology, function, and vulnerability. Accordingly, several studies focused on identifying heterogeneous gene expression across DA neuron clusters. Here we review the importance of understanding DA neuron heterogeneity at the molecular level, previous studies detailing heterogeneous gene expression in DA neurons, and finally recent work which brings together previous heterogeneous gene expression profiles in a coordinated manner, at single cell resolution.
... Our western blot analysis suggests that prolonged consumption of a HFD does not affect total striatal DAT protein, but instead reduces the integration of the non-glycosylated 50 kDa DAT isoform into synaptosomal membranes. While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. ...
... Our western blot analysis suggests that prolonged consumption of a HFD does not affect total striatal DAT protein, but instead reduces the integration of the non-glycosylated 50 kDa DAT isoform into synaptosomal membranes. While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. ...
... While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. Taken together, these studies suggest that the decreased membrane levels of 50 kDa DAT could contribute to the reuptake deficit we observed in 6 wk HFD rats. ...
... This confirms previous detailed studies on this transporter in the adult human SNC. 36 ...
... As previously described, 35,36 DAT (glycosylated and nonglycosylated) and VMAT are also localized to these dopamine neurons. Subregional differences in expression were observed for D2 and D1 dopamine receptors and glycosylated and total DAT and VMAT. ...
... Previous studies have shown reductions in DAT mRNA with aging 55 but others have shown preservation of the proteins involved in dopamine biosynthesis and compartmentation. 56 Importantly, we found a similar distribution of neurons expressing glycosylated DAT protein to that recently published, 36 with higher levels of the active form of DAT in the ventral SNC dopamine neurons, which are more vulnerable to PD. We have demonstrated that DAT expression and DAT glycosylation is highly variable within the midbrain dopamine cell groups. ...
Parkinson's disease (PD) patients have increased susceptibility to impulse control disorders. Recent studies have suggested that alterations in dopamine receptors in the midbrain underlie impulsive behaviors and that more impulsive individuals, including patients with PD, exhibit increased occupancy of their midbrain dopamine receptors. The cellular location of dopamine receptor subtypes and transporters within the human midbrain may therefore have important implications for the development of impulse control disorders in PD. The localization of the dopamine receptors (D1-D5) and dopamine transporter proteins in the upper brain stems of elderly adult humans (n = 8) was assessed using single immunoperoxidase and double immunofluorescence (with tyrosine hydroxylase to identify dopamine neurons). The relative amount of protein expressed in dopamine neurons from different regions was assessed by comparing their relative immunofluorescent intensities. The midbrain dopamine regions associated with impulsivity (medial nigra and ventral tegmental area [VTA]) expressed less dopamine transporter on their neurons than other midbrain dopamine regions. Medial nigral dopamine neurons expressed significantly greater amounts of D1 and D2 receptors and vesicular monoamine transporter than VTA dopamine neurons. The heterogeneous pattern of dopamine receptors and transporters in the human midbrain suggests that the effects of dopamine and dopamine agonists are likely to be nonuniform. The expression of excitatory D1 receptors on nigral dopamine neurons in midbrain regions associated with impulsivity, and their variable loss as seen in PD, may be of particular interest for impulse control. © 2013 Movement Disorder Society.
... Our western blot analysis suggests that prolonged consumption of a HFD does not affect total striatal DAT protein, but instead reduces the integration of the non-glycosylated 50 kDa DAT isoform into synaptosomal membranes. While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. ...
... Our western blot analysis suggests that prolonged consumption of a HFD does not affect total striatal DAT protein, but instead reduces the integration of the non-glycosylated 50 kDa DAT isoform into synaptosomal membranes. While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. ...
... While DAT glycosylation improves the rate of dopamine transport and increases membrane surface stability [45,46,47], non-glycosylated DAT from humans [45,46] as well as rats [47] readily transports dopamine. Additionally, immunolabeling experiments reveal that levels of non-glycosylated DAT are higher in the ventral compared to dorsal striatum in both monkeys and humans [47]. Taken together, these studies suggest that the decreased membrane levels of 50 kDa DAT could contribute to the reuptake deficit we observed in 6 wk HFD rats. ...
The development of diet-induced obesity (DIO) can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT) expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD) or low (LFD) fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA) and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO.
... Protein glycosylation, the enzymatic addition of N-linked or O-linked oligosaccharide moieties to glycoproteins, is one of the most common posttranslational modifications of many functional proteins, and has a profound effect on numerous biological processes including cell-cell interactions, cell signal transduction, subcellular and extracellular trafficking, etc. The oligosaccharide moieties of glycoprotein also affect protein structures via contributing to protein conformation, stability, folding, and solubility [7][8][9][10]. Glycosylation plays particularly crucial roles in central nervous system (CNS) too [10]. ...
... Glycosylation plays particularly crucial roles in central nervous system (CNS) too [10]. For example, DAT (dopamine transporter), an N-glycosylated protein, has been demonstrated that the N-glycosylated DAT transports DA more efficiently than its non-glycosylated form which is related to PD [7]. Abnormal glycosylation has major implications for human health. ...
Protein glycosylation plays a crucial role in central nervous system, and abnormal glycosylation has major implications for human diseases. This study aims to evaluate an etiological implication of the variation in glycosylation for Parkinson’s disease (PD), a neurodegenerative disorder. Based on a PD mouse model constructed by the intraperitoneal injection with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, glycosylation variation was accessed using biotinylated lectin of dolichos biflorus agglutinin (DBA) specific for the exposed N-acetylgalactosamine linked to glycoprotein. Consequently, a glycoprotein with a significantly reduced N-acetylgalactosamination was identified as ADP/ATP translocase 1 (ANT1) by lectin affinity chromatography coupled with MALDI-TOF MS/MS (mass spectrometry), and confirmed by the analysis of dual co-immunofluorescence and Western blot. A tissue-specific distribution of de-N-acetylgalactosaminated ANT1 was found to be correlated with high risk of PD. At cellular level, an obvious co-aggregation between ANT1 and DBA was only found in the MPP⁺-induced PD-like cell model using dual co-immunofluorescence. Thus, we found that ANT1 was a potential glycoprotein with terminal N-acetylgalactosamine moiety, and the variation of glycosylation in ANT1 was associated with PD. This investigation provides an innovative insight in protein glycosylation with PD pathogenesis.
... data into stereotaxic space, structural MRI data were acquired on a 3T Siemens Tim Trio system (Siemens Medical Systems, Iselin, N.J.) equipped with 32-channels. High-resolution structural data were acquired using a T1-weighted magnetization-prepared rapid acquisition with gradient multi echo For the PET scan, (approximately) 10 mCi of [ 11 C]altropane was injected intravenously over [20][21][22][23][24][25][26][27][28][29][30] sec. Images were acquired over 60 minutes in 39 frames of increasing duration (8 frames of 15 sec, 4 frames of 60 sec, 27 frames of 120 sec). ...
... glycosylated forms of DAT (50, 60/65 and 80kDa). The 80 kDa DAT is recognized as the mature form of the molecule28,29 .Post-mortem analyses of nucleus accumbens DATIn the in vivo PET analyses, groups did not differ in BPND in the nucleus accumbens.To further evaluate the role of the nucleus accumbens in MDD, we performed analyses probing DAT expression in this region. Relative to healthy controls, the MDD group had significantly lower expression of the immature (non-glycosylated) form of DAT (40 kDa) (F(1,25)=4.69, ...
Importance
Major depressive disorder (MDD) might involve dopamine (DA) reductions. The DA transporter (DAT) regulates DA clearance and neurotransmission and is sensitive to DA levels, with preclinical studies (including those involving inescapable stressors) showing that DAT density decreases when DA signaling is reduced. Despite preclinical data, evidence of reduced DAT in MDD is inconclusive.
Objective
Using a highly selective DAT positron emission tomography (PET) tracer ([¹¹C] altropane), DAT availability was probed in individuals with MDD who were not taking medication. Levels of DAT expression were also evaluated in postmortem tissues from donors with MDD who died by suicide.
Design, Setting, and Participants
This cross-sectional PET study was conducted at McLean Hospital (Belmont, Massachusetts) and Massachusetts General Hospital (Boston) and enrolled consecutive individuals with MDD who were not taking medication and demographically matched healthy controls between January 2012 and March 2014. Brain tissues were obtained from the Douglas-Bell Canada Brain Bank. For the PET component, 25 individuals with current MDD who were not taking medication and 23 healthy controls recruited from McLean Hospital were included (all provided usable data). For the postmortem component, 15 individuals with depression and 14 healthy controls were considered.
Intervention
PET scan.
Main Outcomes and Measures
Striatal and midbrain DAT binding potential was assessed. For the postmortem component, tyrosine hydroxylase and DAT levels were evaluated using Western blots.
Results
Compared with 23 healthy controls (13 women [56.5%]; mean [SD] age, 26.49 [7.26] years), 25 individuals with MDD (19 women [76.0%]; mean [SD] age, 26.52 [5.92] years) showed significantly lower in vivo DAT availability in the bilateral putamen and ventral tegmental area (Cohen d range, −0.62 to −0.71), and both reductions were exacerbated with increasing numbers of depressive episodes. Unlike healthy controls, the MDD group failed to show an age-associated reduction in striatal DAT availability, with young individuals with MDD being indistinguishable from older healthy controls. Moreover, DAT availability in the ventral tegmental area was lowest in individuals with MDD who reported feeling trapped in stressful circumstances. Lower DAT levels (and tyrosine hydroxylase) in the putamen of MDD compared with healthy controls were replicated in postmortem analyses (Cohen d range, −0.92 to −1.15).
Conclusions and Relevance
Major depressive disorder, particularly with recurring episodes, is characterized by decreased striatal DAT expression, which might reflect a compensatory downregulation due to low DA signaling within mesolimbic pathways.
... Otherwise, when this balance is lost, it can lead to the settlement of OS causing damage to proteins, lipids and DNA, which may impair the neurotransmission of different brain areas [68]. Considering that oxidative metabolism is found to be impaired in addiction, and the potential of DA auto-oxidation [69], along with MOD antioxidant properties [25], we decided to assess some aspects of the oxidative status of our animals. ...
... Considering the potential of DA to affect the dopaminergic system, an exacerbated increase in the dopaminergic activity is a source of OS in the brain [69]. This event was observed in animals that received AMPH, and it was counteracted by MOD administration. ...
Addiction is a serious health problem which leads to general social impairment. The period of adolescence plays a significant role in drug abuse liability. Psychostimulants, such as modafinil (MOD), are majorly used by teenagers seeking improvements in cognition, which contributes to its indiscriminate use. The present study aimed to investigate the influence of MOD (64 mg/kg by gavage, once a day) treatment during adolescence (PND 28-42) on amphetamine (AMPH, 4 mg/kg i.p.) conditioned place preference (CPP) in early adulthood (PND 60). Our findings showed that AMPH increased CPP for the drug and anxiety-like behaviours; on the other hand, AMPH decreased the number of crossings and recognition index. In addition, AMPH decreased catalase activity and increased reactive species, malondialdehyde and carbonyl protein levels in the hippocampus. AMPH also increased pro-BDNF, Trk-B, DAT, D1R and decreased BDNF and D2R immunoreactivity. Contrarily, animals pre-exposed to MOD showed reduced AMPH-CPP, no locomotor impairment, less anxiety-like behaviours and no memory deficits. In addition, MOD showed antioxidant activity by preventing AMPH-induced oxidative damage in the hippocampus. Moreover, molecular analysis showed that MOD was able to modulate the hippocampal dopaminergic system, thus preventing AMPH-induced impairments. Animals that received MOD during adolescence showed reduced AMPH-CPP in early adulthood. These unexpected behavioural effects of MOD on CPP could be due to its hippocampal dopaminergic system modulation, mainly by its action on D1R, which is closely linked to drug addiction. Nevertheless, further studies are necessary. This article is protected by copyright. All rights reserved.
... Taking into account that oxidative stress plays a central role in the pathogenesis of PD, that DA metabolism is the main source of reactive oxygen species in DA-cells, and that the cytosolic content in DA depends mostly on the DA uptake (Bannon, 2005), DAT activity has been involved in DA-cell degeneration in PD. This idea is supported on the fact that midbrain DA-cells showing high susceptibility to degeneration contain higher levels of DAT mRNA and glycosylated (functional) DAT protein than those showing resistance (Uhl et al., 1994;Afonso-Oramas et al., 2009). Moreover, DAT transports natural and synthetic DA analogue neurotoxins, and its blockade or deficient expression makes DA-cells resistant to these neurotoxins (Bezard et al., 1999;Schober, 2004;Afonso-Oramas et al., 2010). ...
... For example, DAT activity is exacerbated in the striatum of GDNF heterocygous mice (Boger et al., 2007;Littrell et al., 2012). In addition, data from our group show that constitutive GDNF mRNA levels in the ventral striatum are higher than in the dorsal striatum, while DAT protein levels and activity are higher in the dorsal striatum than in the ventral striatum (Barroso-Chinea et al., 2005;Afonso-Oramas et al., 2009). In this respect, Zhu and coworkers (2015) have recently showed that the Rho-family guanine nucleotide exchange factor protein Vav2 interacts with the GDNF receptor tyrosine kinase Ret, forming a functional complex that downregulates DAT activity. ...
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.
... In the following decade numerous studies have established that the motor symptoms are attributed to the loss of MbDNs in the SNpc and the decline of DA in the striatum, which are responsible for most, if not all, motor symptoms (Fearnley and Lees, Introduction vitro studies demonstrated that DAT activity depends on its glycosylation status, with the glycosylated DAT form transporting DA more efficiently than the non-glycosylated form (Torres et al., 2003;Li et al., 2004). Interestingly, it has been shown that somata and terminals of the nigrostriatal compartment (ventrocaudal SNpc and dorsal striatum) have higher expression levels of glyco-DAT than those of the rostromedial SNpc (Afonso- Oramas et al., 2009). In PD MbDNs located in ventrolateral and caudal region of the SNpc are more vulnerable than those in the rostromedial and dorsal region (German et al., 1989;Damier et al., 1999), suggesting that differences in DAT post-transcriptional regulation may be involved in the differential vulnerability of MbDNs (Gonzales- Hernandez et al., 2004). ...
... To quantify the density of the projections, the intensity of TH and glyco-DAT fluorescence in the striatum or the number of pixels in the amygdala and PFC were measured in wildtype and Gli2 ΔMb>E9.0 mutant brains (Figure 18B-K). Glyco-DAT is expressed at high levels in MbDN projections to the striatum, but only weakly in projections to the PFC or amygdala(Afonso-Oramas et al., 2009). Surprisingly, despite the significant reduction in the number of SNpc and VTA MbDNs in Gli2 ΔMb>E9.0 brains(Figure 16E), there was no significant difference in the fluorescent intensity of TH or glyco-DAT staining in the CPu complex of the mutants compared to control brains (Figure 18B-E). ...
Midbrain dopaminergic neurons (MbDNs) in the ventral tegmental area (VTA) and substantia
nigra pars compacta (SNpc) modulate cognition, reward behavior and voluntary movement,
respectively. Recent findings indicate that VTA and SNpc MbDNs form subpopulations that
are divergent in their electrophysiological features, functions and vulnerability to
neurodegeneration in Parkinson’s disease. This diversity can be correlated with the
anatomical organization of these two populations and their afferent and efferent connections.
However, it is largely unexplored how MbDN diversity is established during development.
Previous studies have demonstrated that the identity of MbDN subtypes can be directly linked
to their temporal and spatial origin in the embryonic midbrain (Blaess et al., 2011; Hayes et
al., 2011). Different subsets of MbDNs are derived from a ventral progenitor pool in the
developing midbrain that is subdivided into a medial and a lateral domain. The relationship
between developmental origin and the identity of MbDNs in the adult brain is likely reflected
by the regulated activity of genes inducing cell fate during embryogenesis. Thus, the timing of
Sonic hedgehog (Shh) signaling might play a role in the determination of the fate of MbDN
subpopulations, since MbDN precursors respond differently to Shh.
To address whether Shh signaling regulates the specification of MbDN subtypes, conditional
gene inactivation approach was used in this study. Removal of Shh signaling at particular
time point during MbDN induction results in the selective loss of a specific subset of MbDN
precursors in the embryo. Using viral tracing and immunohistochemical analysis, this study
demonstrates that this population of MbDN precursors gives rise to mesocortical projection
neurons in the VTA. Furthermore, optogenetics and physiological analysis reveals that
mesocortical MbDNs inhibit prefrontal cortical pyramidal neurons via an inhibitory cortical
microcircuit. Other MbDN-derived projections are largely unaffected. Thus, temporally
precise Shh signaling in the midbrain is required for establishing a specific mesocortical
microcircuit. This is the first study establishing a causal link between early developmental
induction mechanisms and the functional properties of MbDNs in the adult brain.
Furthermore, constitutive activation of Shh signaling results in a massive increase in the
number of MbDNs and the ventralization of the dorsal midbrain. Interestingly, analysis of
MbDNp domain shows that only medial MbDN precursor domain was significantly increased.
Due to the perinatal lethality of the mutant mice, investigation of MbDN specification in the
adult brains was not possible.
In addition, this study demonstrates that the development of the red nucleus (RN) neuron
subpopulations is determined by the duration of Shh signaling as well. While inactivation of
Shh signaling does not affect the generation of the parvocellular RN neurons, the neurons of
magnocellular RN are severely reduced and disorganized.
... Drd2 expression itself was not affected in degenerating Rgs6 2/2 mDA neurons (Fig. 8A). However, the vSNc TH low neurons showed evidence of increased DA signaling, namely accumulation of phospho-Erk1/2 (Fig. 8B) and enhanced glycosylated dopamine transporter (Slc6a3/DAT) expression in the mutant (Fig. 8C), consistent with a putative loss of negative Rgs6 input on dopamine signaling [50]. Since the Pitx3 2/2 midbrain exhibits decreased DAT and Drd2 [19], the observed increase in DAT together with phospho-Erk1/2 are consistent with a primary action of Rgs6 inactivation on DA signaling. ...
... Since the Pitx3 2/2 midbrain exhibits decreased DAT and Drd2 [19], the observed increase in DAT together with phospho-Erk1/2 are consistent with a primary action of Rgs6 inactivation on DA signaling. Rgs6 may thus contribute to the auto-regulatory negative feedback of the dopaminergic system and its absence may lead to dopaminedependent oxidative stress and neuronal loss [2,50]. The enhanced phospho-p27 Kip1 and FluoroJade staining support the interpretation that these cells are under stress. ...
Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.
... This procedure was performed according to Afonso-Oramas et al. (2009). The mouse striata were dissected in ice from freshly obtained brains using a brain blocker. ...
... Several commercial anti-DAT antibodies against different fragments of human and rat DAT have been tested in our laboratory. Brain samples of different mammals, including humans and mice, and cells transfected with wild type and mutated DAT forms, were subjected to immunohistochemistry, western-blot and immunoprecipitation (see Afonso-Oramas et al., 2009, 2010Cruz-Muros et al., 2009). These experiments were used to determine their specificity and sensitivity, as well as which band (glycosylated, 75 kDa; partially glycosylated, 68 kDa, and non-glycosylated, 50 kDa) is detected by each of them . ...
... Nevertheless, it must be pointed out that while some studies have reported that inhibition of DAT protects against paraquat-induced toxicity, suggesting a role for DAT in the cellular uptake of paraquat (59), other studies report that paraquat is not a substrate of DAT (60). The vulnerability of distinct subgroups of DAergic neurons observed in PD is directly correlated with basal levels of DAT protein expression and, in particular, with the glycosylated form of DAT (61). For example, DAergic neurons of the SN express higher levels of glycol-DAT, transport DA more efficiently, and are more affected than ventral tegmental neurons (61). ...
... The vulnerability of distinct subgroups of DAergic neurons observed in PD is directly correlated with basal levels of DAT protein expression and, in particular, with the glycosylated form of DAT (61). For example, DAergic neurons of the SN express higher levels of glycol-DAT, transport DA more efficiently, and are more affected than ventral tegmental neurons (61). Furthermore, the DAergic neurons surviving in PD midbrain at time of death exhibit lower DAT gene expression per cell than the DAergic cells in control subjects (62). ...
Parkinson's disease is the second most common neurodegenerative disorder and, at present, has no cure. Both environmental and genetic factors have been implicated in the etiology of the disease; however, the pathogenic pathways leading to neuronal degeneration are still unclear. Parkinson's disease is characterized by the preferential death of a subset of neurons in the mesencephalon that use dopamine as neurotransmitter for synaptic communication. Dopamine is a highly reactive molecule that can lead to cytotoxicity if not properly stored and metabolized. Targeting any of the pathways that tightly control this neurotransmitter holds great therapeutic expectations. In this article we present a comprehensive overview of the cellular pathways that control dopamine fate and discuss potential therapeutic approaches to counteract or slow Parkinson's disease onset and progression.-Bisaglia, M., Greggio, E., Beltramini, M., Bubacco, L. Dysfunction of dopamine homeostasis: clues in the hunt for novel Parkinson's disease therapies.
... It has been found that in the striatum (Str) of PD rat models treated with 6-hydroxydopamine (6-OHDA) and rDAT-HEK cell models exposed to 1methyl-4-phenylpyridinium iodide (MPP + ), non-glycosylated DAT accumulates on the endoplasmic reticulum and Golgi, while glycosylated DAT decreases on both the endoplasmic reticulum and Golgi, as well as on the plasma membrane. It has been suggested that the lack of glycosylation modi cation of DAT may impede its transfer to the plasma membrane in DA neurons [11,12] . The aberrant function of DAT can lead to DA disorders, which may precipitate PD, schizophrenia, attention de cit hyperactivity disorder and other central nervous system pathologies [9,[13][14][15] . ...
The core of clinic treatment of Parkinson's disease (PD) is to enhance dopamine (DA) signaling within the brain. The regulation of dopamine transporter (DAT) is integral to this process. This study aims to explore the regulatory mechanism of glial cell line-derived neurotrophic factor (GDNF) on DAT, thereby gaining a profound understanding its potential value in treating PD. Here, we investigated the effects of GDNF on both cells and mice with PD, including the glycosylation and membrane distribution of DAT detected by immunofluorescence and immunoblotting, DA signal measured by neurotransmitter fiber imaging technology, Golgi morphology observed by electron microscopic, as well as cognitive ability assessed by behavior tests. In this study, GDNF enhanced the glycosylation and membrane distribution of DAT of the injured DA neurons both in vivo and invitro, while reversing GRASP65 loss and Golgi fragmentation leading to alleviation of DAT accumulation in the Golgi. Moreover, GRASP65 overexpression increased DAT distribution in PD cells and mice, whereas, the inhibition of GRASP65 could leading to diminished role of GDNF on DAT. In addition, GDNF could enhance the reutilization of neurotransmitter uptake by presynaptic membranes in the PFC and enhance the effective DA release signal after a single electrical stimulation, ultimately improving the cognitive impairment of PD mice. Thus, we proposed that GDNF promotes the glycosylation and membrane distribution of DAT by facilitating Golgi reaggregation, leading to an enhancement of DA signal utilization, ultimately resulting in an improvement in cognitive ability of PD mice. This study illustrates from a new perspective the beneficial role of GDNF in enhancing DA utilization and improving cognition in PD.
... An alternative explanation could be a compensatory downregulation of DAT mRNA levels in the low-expressing neurons in PD. However, this alternative scenario is less likely since the same differential expression of DAT mRNA was documented in controls across species (Sanghera et al., 1994;Haber et al., 1995;Bannon and Whitty, 1997;Afonso-Oramas et al., 2009). Along the same lines, a recent meta-analysis study suggests that the SLC6A3 10R variant, associated with relatively lower expression activity of DAT, may be a protective factor in susceptibility to PD (Zeng et al., 2021). ...
One of the hallmarks of Parkinson’s disease (PD) is the progressive loss of dopaminergic neurons and associated dopamine depletion. Several mechanisms, previously considered in isolation, have been proposed to contribute to the pathophysiology of dopaminergic degeneration: dopamine oxidation-mediated neurotoxicity, high dopamine transporter (DAT) expression density per neuron, and autophagy-lysosome pathway (ALP) dysfunction. However, the interrelationships among these mechanisms remained unclear. Our recent research bridges this gap, recognizing autophagy as a novel dopamine homeostasis regulator, unifying these concepts. I propose that autophagy modulates dopamine reuptake by selectively degrading DAT. In PD, ALP dysfunction could increase DAT density per neuron, and enhance dopamine reuptake, oxidation, and neurotoxicity, potentially contributing to the progressive loss of dopaminergic neurons. This integrated understanding may provide a more comprehensive view of aspects of PD pathophysiology and opens new avenues for therapeutic interventions.
... Partially or non-glycosylated DATs are preferentially endocytosed compared to fully glycosylated DATs (Torres et al., 2003;Li et al., 2004) and N-glycosylation of DAT may facilitate its oligomerization in relation to its intracellular trafficking (Torres et al., 2003). Furthermore, glycosylation increases DAT's susceptibility to bind to various drugs such as cocaine (Li et al., 2004) and reduced glycosylation of DAT in the human striatum and midbrain leads to increased PD susceptibility (Afonso-Oramas et al., 2009). These studies validate DAT as a therapeutic target and provide avenues for pharmacological manipulation of DAT to reduce disease severity. ...
The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na+/Cl− dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson’s disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.
... Although these techniques do not have the same cellular and molecular resolution as more recent scRNA-seq approaches, these data suggested that also the human mDA system can be divided into different neuron subtypes characterized by the expression of specific molecular markers. Mapping of immunohistochemical expression data of marker genes (CALB1, PITX3, DCC and GIRK2, which encodes G protein-gated inwardly rectifying potassium channel 2 and is also known as KCNJ6) 9,48,49 onto the human midbrain revealed molecularly distinct neuron subtypes that in part are similar to those described in the mouse 9,43,[50][51][52] (Fig. 1c). For example, the expression pattern of ALDH1A1, SOX6 and CALB1 in the human midbrain suggests that humans and mice have similar neuron subtypes in the SNc (subtypes 1 and 2 in mice) (Fig. 1a,c). ...
The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviours and are linked to various brain diseases. Considerable progress has been made in identifying mDA neuron subtypes, and recent work has begun to unveil how these neuronal subtypes develop and organize into functional brain structures. This progress is important for further understanding the disparate physiological functions of mDA neurons and their selective vulnerability in disease, and will ultimately accelerate therapy development. This Review discusses recent advances in our understanding of molecularly defined mDA neuron subtypes and their circuits, ranging from early developmental events, such as neuron migration and axon guidance, to their wiring and function, and future implications for therapeutic strategies. Recent technological advances have provided insights into the diversity of neuronal subtypes within the midbrain dopamine system. In this Review, Garritsen and colleagues discuss molecular and functional distinctions between subtypes and describe mechanisms underlying their development, wiring and function.
... The N-glycosylation of DAT plays a role in the vulnerability of dopaminergic cells in PD. DAT transporter expression and function are regulated by N-glycosylation [77]. DAT is highly expressed in the SN, and translocating excess DA regulates the levels and effects of DA. ...
Parkinson’s disease (PD) is a neurodegenerative disorder that affects over 10 million aging people worldwide. This condition is characterized by the degeneration of dopaminergic neurons in the pars compacta region of the substantia nigra (SNpc) and by aggregation of proteins, commonly α-synuclein (SNCA). The formation of Lewy bodies that encapsulate aggregated proteins in lipid vesicles is a hallmark of PD. Glycosylation of proteins and neuroinflammation are involved in the pathogenesis. SNCA has many posttranslational modifications and interacts with components of membranes that affect aggregation. The large membrane lipid dolichol accumulates in the brain upon age and has a significant effect on membrane structure. The replacement of dopamine and dopaminergic neurons are at the forefront of therapeutic development. This review examines the role of membrane lipids, glycolipids, glycoproteins and dopamine in the aggregation of SNCA and development of PD. We discuss the SNCA-dopamine-neuromelanin-dolichol axis and the role of membranes in neuronal stem cells that could be a regenerative therapy for PD patients.
... Moreover, α-synuclein aggregation affects posttranslational modification of other proteins, including glycosylation [30]. Specific glycosylation of dopamine transporters reported to occur in nigrostriatal dopaminergic neurons was found to be associated with the same degenerated cells in PD patients [31] (Table 3). ...
Neuronal homeostasis depends on both simple and complex sugars (the glycoconjugates), and derangement of their metabolism is liable to impair neural function and lead to neurodegeneration. Glucose levels boost glycation phenomena, a wide series of non-enzymatic reactions that give rise to various intermediates and end-products that are potentially dangerous in neurons. Glycoconjugates, including glycoproteins, glycolipids, and glycosaminoglycans, contribute to the constitution of the unique features of neuron membranes and extracellular matrix in the nervous system. Glycosylation defects are indeed frequently associated with nervous system disturbances and neurodegeneration. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms associated with the loss of dopaminergic neurons in the pars compacta of the substantia nigra. Neurons present intracytoplasmic inclusions of α-synuclein aggregates involved in the disease pathogenesis together with the impairment of the autophagy-lysosome function, oxidative stress, and defective traffic and turnover of membrane components. In the present review, we selected relevant recent contributions concerning the direct involvement of glycation and glycosylation in α-synuclein stability, impaired autophagy and lysosomal function in PD, focusing on potential models of PD pathogenesis provided by genetic variants of glycosphingolipid processing enzymes, especially glucocerebrosidase (GBA). Moreover, we collected data aimed at defining the glycomic profile of PD patients as a tool to help in diagnosis and patient subtyping, as well as those pointing to sugar-related compounds with potential therapeutic applications in PD.
... Parkinson's disease rates [35]. It is also important to note that balance between types of modifications dictates transport capability, therein suggesting a more complex regulatory framework instilled by posttranslational modifications [32]. ...
The dopamine transporter (DAT) is a membrane protein essential to dopamine homeostasis. Abnormal dopamine homeostasis is implicated in multiple conditions including the newly discovered dopamine transporter deficiency syndrome (DTDS). DTDS is an autosomal-recessive disorder caused by mutations that impair DAT maturation. Bupropion and ibogaine can rescue DTDS DAT mutants. We therefore examine the structure-activity relationships responsible, in hopes of discovering more efficacious and potent compounds. An SAR-based screen of bupropion and ibogaine analogs demonstrated necessity for a secondary amine and single halogen substitution on bupropion along with a flexible heterocyclic ring, chair-like substituent and hydroxyl substitution on ibogaine. Lead candidates noribogaine and PAL594 were able to rescue mature DAT. We also report that there is no link between DAT inhibition and chaperoning, suggesting that chaperoning is mediated allosterically. Future studies should aim to validate the presence of the putative DAT allosteric site, computer-assisted drug discovery and a mouse model.
... Additionally, alterations in the expression of VMAT2, DAT, and ALDH1A within the SNc of RGS6 -/mice may reveal the toxic mechanism responsible for SNc DA neuron degeneration as previous studies using transgenic mice have demonstrated that while VMAT2 and ALDH1A protect SNc DA neurons and oppose PD, increases in DAT expression provoke SNc neurodegeneration (66)(67)(68)(69)(70)(71). Exactly how alteration in these factors lead to neurodegeneration is uncertain, but it is possible that the dysfunction in the transportation, storage and catabolism of DA caused by their altered expression may contribute to an accumulation of cytotoxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL) in the cytosol (Figure 7) (72)(73)(74)(75)(76). ...
Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.
... d Some FLAG tagged. -Oramas et al., 2009;Carvelli et al., 2002;Foster and Vaughan, 2011;Hong and Amara, 2010;Jones et al., 2017;Moritz et al., 2013;Morón et al., 2003;Richards and Zahniser, 2009;Vaughan et al., 1997;Zahniser et al., 1999) VTA Slices 0.33 ± 0.03 a (Mebel et al., 2012) Striatum (in vivo) 0.7 a 10-12 (K T ) ( Sabeti et al., 2002;Zahniser et al., 1999) Mouse Striatal Synaptosomes 38 ± 7 0.06 ± 0.01 (Rao et al., 2013) NAc Slices 1.0 a 0.2 (Lu et al., 2015) Human Striatal Synaptosomes 0.41 (Mash et al., 2002) Drosophila Channelrhodospin2 (ChR2) DA neurons 0.11 ± 0.02 a 1.3 ± 0.6 (Vickrey et al., 2013) a μM/s. (Eshleman et al., 1995;Kitayama et al., 1992) hDAT LLC-PK 1 2000 ± 200 22 ± 3 (Zhen and Reith, 2016) CHO 5-10 b 8-9 ( Midde et al., 2015;Slusher et al., 1997) HEK 1000-13,000 16-22 (Berfield et al., 1999;Chen et al., 2000;Wang et al., 2003;Zhen et al., 2015Zhen et al., , 2005) N2a 2500 ± 300 19-22 (Little et al., 2002;Zhang et al., 1998) differences, as velocity depends on surface expression of the transporter, which can vary greatly between systems and studies. ...
The dopamine transporter (DAT) plays a critical role in dopamine (DA) homeostasis by clearing transmitter from the extraneuronal space after vesicular release. DAT serves as a site of action for a variety of addictive and therapeutic reuptake inhibitors, and transport dysfunction is associated with transmitter imbalances in disorders such as schizophrenia, attention deficit hyperactive disorder, bipolar disorder, and Parkinson disease. In this review, we describe some of the model systems that have been used for in vitro analyses of DAT structure, function and regulation, and discuss a potential relationship between transporter kinetic values and membrane cholesterol.
... Interestingly, co-immunoprecipitation experiments between snapin and DAT in the striatum suggest the involvement of the immature form of DAT, similar to our findings [55]. The interaction with the non-glycosylated form of DAT is interesting as DAT activity depends not only on its membrane expression but also on its glycosylation status as shown in vitro and by the differential vulnerability of midbrain DA cells in Parkinson's disease [56]. ...
Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.
... In this study, we measured the expression of glycosylated and nonglycosylated DAT in NAcc and Striatum. The antibody used in this work has been used previously detecting 2 specific bands corresponding to glycosylated (80 KDa) and non-glycosylated (69 KDa) DAT expression [35]. The DAT protein undergoes post-translational modifications in the Golgi apparatus [36] and glycosylation is an important step for the trafficking of DAT to the plasma membrane [36]. ...
Research in programming is focused on the study of stimuli that alters sensitive periods in development, such as prenatal and neonatal stages, that can produce long-term deleterious effects. These effects can occur in various organs or tissues such as the brain, affecting brain circuits and related behaviors. Our laboratory has demonstrated that neonatal programming with sex hormones affects the mesocorticolimbic circuitry, increasing the synthesis and release of dopamine (DA) in striatum and nucleus accumbens (NAcc). However, the behavioral response to psychostimulant drugs such as methylphenidate and the possible mechanism(s) involved have not been studied in adult rats exposed to sex hormones during the first hours of life. Thus, the aim of this study was to examine the locomotor activity induced by methylphenidate (5mg/kg i.p.) and the expression of the DA transporter (DAT) in NAcc of adult rats exposed to a single dose of testosterone propionate (TP: 1mg/50μLs.c.) or estradiol valerate (EV: 0.1mg/50μLs.c.) at postnatal day 1. Our results demonstrated that adult female rats treated with TP have a lower methylphenidate-induced locomotor activity compared to control and EV-treated adult female rats. This reduction in locomotor activity is related with a lower NAcc DAT expression. However, neither methylphenidate-induced locomotor activity nor NAcc DAT expression was affected in EV or TP-treated adult male rats. Our results suggest that early exposure to sex hormones affects long-term dopaminergic brain areas involved in the response to psychostimulants, which could be a vulnerability factor to favor the escalating doses of drugs of abuse.
... However, the expression of a few genes is commonly used to molecularly distinguish larger mDA domains. For example, the glycosylated active form of the dopamine transporter (glyco-DAT) and the G-protein-gated inwardly rectifying K + channel (Girk2) are more abundantly expressed by SNc and dorso-lateral VTA mDA neurons (Schein et al., 1998;Thompson et al., 2005;Afonso-Oramas et al., 2009), while Calbindin 1 (Calb1) expression is enriched in mDA neurons of the VTA and of the dorsal tier of the SNc Di Salvio et al., 2010;Fu et al., 2012). Within the VTA, the transcription factor Otx2 strongly labels ventro-medial mDA neurons and gradually deceases in the central and dorso-lateral VTA (Simeone et al., 2011). ...
The midbrain dopamine (mDA) system is involved in the control of cognitive and motor behaviors, and is associated with several psychiatric and neurodegenerative diseases. mDA neurons receive diverse afferent inputs and establish efferent connections with many brain areas. Recent studies have unveiled a high level of molecular and cellular heterogeneity within the mDA system with specific subsets of mDA neurons displaying select molecular profiles and connectivity patterns. During mDA neuron development, molecular differences between mDA neuron subsets allow the establishment of subset-specific afferent and efferent connections and functional roles. In this review, we summarize and discuss recent work defining novel mDA neuron subsets based on specific molecular signatures. Then, molecular cues are highlighted that control mDA neuron migration during embryonic development and that facilitate the formation of selective patterns of efferent connections. The review focuses largely on studies that show differences in these mechanisms between different subsets of mDA neurons and for which in vivo data is available, and is concluded by a section that discusses open questions and provides directions for further research.
... For example, post-transcriptional modifications that regulate the activity of specific gene products might differ between different populations of DA neurons. One prominent example is the glycosylation of Slc6a3 (indicative of the active form of the transporter), the expression of which has been shown to be higher in the SNc than in the VTA (Afonso-Oramas et al. 2009;Di Salvio et al. 2010a). Another possibility is diversity concerning bioenergetic parameters. ...
Dopaminergic neurons in the ventral mesencephalon (the ventral mesencephalic dopaminergic complex) are known for their role in a multitude of behaviors, including cognition, reward, addiction and voluntary movement. Dysfunctions of these neurons are the underlying cause of various neuropsychiatric disorders, such as depression, addiction and schizophrenia. In addition, Parkinson's disease (PD), which is the second most common degenerative disease in developed countries, is characterized by the degeneration of dopaminergic neurons, leading to the core motor symptoms of the disease. However, only a subset of dopaminergic neurons in the ventral mesencephalon is highly vulnerable to the disease process. Indeed, research over several decades revealed that the neurons in the ventral mesencephalic dopaminergic complex do not form a homogeneous group with respect to anatomy, physiology, function, molecular identity or vulnerability/dysfunction in different diseases. Here, we review how the concept of dopaminergic neuron diversity, assisted by the advent and application of new technologies, evolved and was refined over time and how it shaped our understanding of PD pathogenesis. Understanding this diversity of neurons in the ventral mesencephalic dopaminergic complex at all levels is imperative for the development of new and more selective drugs for both PD and various other neuropsychiatric diseases.
image Several decades of research revealed that the neurons in the ventral mesencephalic dopaminergic complex do not form a homogeneous group in respect to anatomy, physiology, function, molecular identity or vulnerability/dysfunction in diseases like Parkinson's disease (PD). Here, we review how this concept evolved and was refined over time and how it shaped our understanding of the pathogenesis of PD. Source of the midbrain image: www.wikimd.org/wiki/index.php/The_Midbrain_or_Mesencephalon ; downloaded 28.01.2016. See also Figures and of the paper.
This article is part of a special issue on Parkinson disease .
... Alternatively, the AB1591P DAT antibody was used on rat tissue with very similar results. This is supported by a recent study in the rat, where the two antibodies were shown to selectively label dopaminergic neurons and terminals, and produce very similar patterns of labeling (79). ...
Significance
In vertebrates, the contribution of dopamine neurons to locomotor control is traditionally attributed to their ascending projections to the basal ganglia that, in turn, project down to brainstem locomotor networks. We recently discovered in lampreys that brainstem networks receive a direct descending dopaminergic input that increases locomotor output. Here, we show that this descending dopaminergic pathway is conserved in higher vertebrates. We found that dopamine is released in salamander brainstem locomotor networks, together with reticulospinal cell activation, known to trigger locomotion. Dopamine is released in rat brainstem locomotor networks, and amphetamine potentiates dopamine release in vivo. Finally, brainstem locomotor networks in human tissue contain dopaminergic terminals. Our findings have important implications for understanding the locomotor role of dopamine in vertebrates.
... In the end, RGS6 deficient neurons would be expected to not only have an impaired ability to synthesize (low TH) DA and package it into vesicles (low VMAT2) but would also increase their DA reuptake (high DAT). Together, these changes could increase cytosolic DA in SNc neurons causing neurodegeneration through accumulation of cytotoxic DA metabolites, such as 3,4dihydroxyphenylacetaldehyde (DOPAL) (82,83) (Fig. 4c). ...
Regulator of G protein signaling (RGS) proteins are gatekeepers regulating the cellular responses induced by G protein-coupled receptor (GPCR)-mediated activation of heterotrimeric G proteins. Specifically, RGS proteins determine the magnitude and duration of GPCR signaling by acting as a GTPase-activating protein for Gα subunits, an activity facilitated by their semiconserved RGS domain. The R7 subfamily of RGS proteins is distinguished by two unique domains, DEP/DHEX and GGL, which mediate membrane targeting and stability of these proteins. RGS6, a member of the R7 subfamily, has been shown to specifically modulate Gαi/o protein activity which is critically important in the central nervous system (CNS) for neuronal responses to a wide array of neurotransmitters. As such, RGS6 has been implicated in several CNS pathologies associated with altered neurotransmission, including the following: alcoholism, anxiety/depression, and Parkinson's disease. In addition, unlike other members of the R7 subfamily, RGS6 has been shown to regulate G protein-independent signaling mechanisms which appear to promote both apoptotic and growth-suppressive pathways that are important in its tumor suppressor function in breast and possibly other tissues. Further highlighting the importance of RGS6 as a target in cancer, RGS6 mediates the chemotherapeutic actions of doxorubicin and blocks reticular activating system (Ras)-induced cellular transformation by promoting degradation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) to prevent its silencing of pro-apoptotic and tumor suppressor genes. Together, these findings demonstrate the critical role of RGS6 in regulating both G protein-dependent CNS pathology and G protein-independent cancer pathology implicating RGS6 as a novel therapeutic target.
... This contention is further supported by the finding that the rectal temperatures were very similar in reserpine-treated wild-type mice and in D 2 receptor knockout mice after METH injection, whereas the neurotoxic effects clearly differed between these two experimental groups (Granado et al., 2011a). Other plausible hyperthermia-independent mechanisms underlying the neuroprotection observed after D 2 receptor inactivation are provided by the evidence that the activity of striatal DAT, which is crucial for METH-induced neurotoxicity (Fumagalli et al., 1998;Manning-Bog et al., 2007;Afonso-Oramas et al., 2009), is reduced in the absence of D 2 receptor (Dickinson et al., 1999). Thus, it is proposed that the decrease in DAT activity produced by inactivation of the D 2 receptor blocks DA reuptake, resulting in lower intra-cytosolic DA levels and hence, neuroprotection (Fig. 6) . ...
Amphetamine-related drugs, such as 3,4-methylenedioxymethamphetamine (MDMA) and metham-
phetamine (METH), are popular recreational psychostimulants. Several preclinical studies have
demonstrated that, besides having the potential for abuse, amphetamine-related drugs may also elicit
neurotoxic and neuroinflammatory effects. The neurotoxic potentials of MDMA and METH to
dopaminergic and serotonergic neurons have been clearly demonstrated in both rodents and nonhuman
primates. This review summarizes the species-specific cellular and molecular mechanisms
involved in MDMA and METH-mediated neurotoxic and neuroinflammatory effects, along with the most
important behavioral changes elicited by these substances in experimental animals and humans.
Emphasis is placed on the neuropsychological and neurological consequences associated with the
neuronal damage. Moreover, we point out the gap in our knowledge and the need for developing
appropriate therapeutic strategies to manage the neurological problems associated with amphetamine-
related drug abuse.
... In addition, because of the role of glycosylation in neurodegenerative diseases, our glyco-SRM approach will likely be applicable to the analysis of Parkinson's disease or Huntington's disease. 44 ...
Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (α64N, α81N and α123N) and three in the beta subunit (β64N, β127N, and β147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n=13) and high hippocampal atrophy (n=14). In our pilot study, the β64N site shows the most significant regulations between clinical groups. Eight β64N glycoforms are significantly reduced in patients with high atrophy compared to those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimer's disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.
... Glycosylation plays an important role in the susceptibility of DAT to the effects of drugs and disease. Cocaine has a greater inhibitory effect on nonglycosylated DAT (Li et al., 2004), and reduced DAT glycosylation within the human striatum and midbrain strongly correlates with greater PD susceptibility (Afonso-Oramas et al., 2009). Maintaining or enhancing DAT glycosylation, perhaps via pharmacological manipulation, may stabilize DAT plasma membrane localization and uptake and may aid in the preservation of DAT function. ...
Dopamine (DA) plays a well recognized role in a variety of physiologic functions such as movement, cognition, mood, and reward. Consequently, many human disorders are due, in part, to dysfunctional dopaminergic systems, including Parkinson's disease, attention deficit hyperactivity disorder, and substance abuse. Drugs that modify the DA system are clinically effective in treating symptoms of these diseases or are involved in their manifestation, implicating DA in their etiology. DA signaling and distribution are primarily modulated by the DA transporter (DAT) and by vesicular monoamine transporter (VMAT)-2, which transport DA into presynaptic terminals and synaptic vesicles, respectively. These transporters are regulated by complex processes such as phosphorylation, protein-protein interactions, and changes in intracellular localization. This review provides an overview of 1) the current understanding of DAT and VMAT2 neurobiology, including discussion of studies ranging from those conducted in vitro to those involving human subjects; 2) the role of these transporters in disease and how these transporters are affected by disease; and 3) and how selected drugs alter the function and expression of these transporters. Understanding the regulatory processes and the pathologic consequences of DAT and VMAT2 dysfunction underlies the evolution of therapeutic development for the treatment of DA-related disorders.
... brains could only be observed in the olfactory tubercle ( Fig. 3b-h,m). Glyco-DAT, which is primarily expressed in MbDN projections to the caudoputamen complex, NAc and olfactory tubercle 28 , was not significantly altered in any of these regions (unpaired t test; CPu, P = 0.2867; NAc, P = 0.3361; OT, P = 0.9048; Supplementary Fig. 5a-c). ...
Midbrain ventral tegmental neurons project to the prefrontal cortex and modulate cognitive functions. Using viral tracing, optogenetics and electrophysiology, we found that mesocortical neurons in the mouse ventrotegmental area provide fast glutamatergic excitation of GABAergic interneurons in the prefrontal cortex and inhibit prefrontal cortical pyramidal neurons in a robust and reliable manner. These mesocortical neurons were derived from a subset of dopaminergic progenitors, which were dependent on prolonged Sonic Hedgehog signaling for their induction. Loss of these progenitors resulted in the loss of the mesocortical inhibitory circuit and an increase in perseverative behavior, whereas mesolimbic and mesostriatal dopaminergic projections, as well as impulsivity and attentional function, were largely spared. Thus, we identified a previously uncharacterized mesocortical circuit contributing to perseverative behaviors and found that the diversity of dopaminergic neurons begins to be established during their progenitor phase.
... While more than 90% of the analyzed SNpc DA neurons and more than 80% of the analyzed VTA DA neurons expressed some level of glycosylated DAT, high levels of glycosylated DAT were found, in particular, in a fraction of DA neurons located in the most vulnerable ventral tier of the SNpc. This is in agreement with the findings of another immunohistochemistry study in rats and human midbrain (Afonso-Oramas et al., 2009). However, ventral tier SNpc DA neurons did not show uniform expression levels of glycosylated DAT, and fewer than 20% of these neurons were characterized by high levels. ...
Numerous disorders of the central nervous system (CNS) are attributed to the selective death of distinct neuronal cell populations. Interestingly, in many of these conditions, a specific subset of neurons is extremely prone to degeneration while other, very similar neurons are less affected or even spared for many years. In Parkinson's disease (PD), the motor manifestations are primarily linked to the selective, progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). In contrast, the very similar DA neurons in the ventral tegmental area (VTA) demonstrate a much lower degree of degeneration. Elucidating the molecular mechanisms underlying the phenomenon of differential DA vulnerability in PD has proven extremely challenging. Moreover, an increasing number of studies demonstrate that considerable molecular and electrophysiologic heterogeneity exists among the DA neurons within the SNpc as well as those within the VTA, adding yet another layer of complexity to the selective DA vulnerability observed in PD. The discovery of key pathways that regulate this differential susceptibility of DA neurons to degeneration holds great potential for the discovery of novel drug targets and the development of promising neuroprotective treatment strategies. This review provides an update on the molecular basis of the differential vulnerability of midbrain DA neurons in PD and highlights the most recent developments in this field.
... Although the mechanism responsible for the preferential loss of DA neurons is still under discussion, a number of neuroprotective mechanisms have been suggested. Although several studies appointed the calcium-binding protein known as calbindin (CB; Gerfen et al., 1985Gerfen et al., , 1987Yamada et al., 1990;Ng et al., 1996;Yuan et al., 2013) as a putative neuroprotective agent, candidates other than CB such as the vesicular monoamine transporter (VMAT2; Liu et al., 1992;Harrington et al., 1996;Miller et al., 1999;Caudle et al., 2007;Afonso-Oramas et al., 2009) have also been suggested. ...
Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus. As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise
... Dopaminergic lesions result in the loss of DAT terminals through degeneration, but there are additional losses in DAT function that result from a simultaneous reduction of DAT export, leading to its accumulation in the ER-Golgi compartment. 13 Also, the ratio of glycosylated to non-glycosylated DAT is elevated in the dopaminergic neuronal populations most affected in PD, 14 suggesting that trafficking of DAT in the neurons comprising the nigrostriatal pathway is especially dependent on efficient ER and Golgi function. Furthermore, pathological excess of α-Syn can trigger ER stress in rats, 15 and similar effects have also been reported recently in a mouse model of synucleinopathy. ...
More than 2 decades of work have yet to conclusively determine the physiological role of the synuclein proteins, even though these abundant brain constituents are participants in a broad array of cellular processes. Among proposed physiological roles is a functional interaction between the synuclein proteins and monoamine transporters contributing to transporter trafficking through direct protein-protein interactions. Recent work shows that an antagonistic effect of the synuclein proteins on the secretory functions of the endoplasmic reticulum and the Golgi apparatus appears to simultaneously influence trafficking of the dopamine transporter and other membrane proteins. Here, we highlight these new findings in view of the broader literature identifying the role of synucleins in protein trafficking and suggest emerging themes for ongoing and future work in the field of synuclein biology.
... The fact that IP-LGF significantly reduces apomorphine-induced rotational behavior and improves motor performance, as assessed by the rotarod test, suggests that LGF modulates the functionality of the DA system in 6-OHDAlesioned rats. In this respect, we should mention that DAT is a protein located on dopaminergic nerve terminals that is involved in DA uptake and is used as a marker for the evaluation of the integrity of the DA system [38][39][40]. As shown here, IP-LGF prevented the decrease in DAT protein expression and immunoreactivity promoted by 6-OHDA in the DA-depleted striatum. ...
Liver growth factor (LGF) is a hepatic mitogen purified some years ago that promotes proliferation of different cell types and the regeneration of damaged tissues, including brain tissue. Considering the possibility that LGF could be used as a therapeutic agent in Parkinson's disease, we analyzed its potential neuroregenerative and/or neuroprotective activity when peripherally administered to unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. For these studies, rats subjected to nigrostriatal lesions were treated intraperitoneally twice a week with LGF (5 microg/rat) for 3 weeks. Animals were sacrificed 4 weeks after the last LGF treatment. The results show that LGF stimulates sprouting of tyrosine hydroxylase-positive terminals and increases tyrosine hydroxylase and dopamine transporter expression, as well as dopamine levels in the denervated striatum of 6-OHDA-lesioned rats. In this structure, LGF activates microglia and raises tumor necrosis factor-alpha protein levels, which have been reported to have a role in neuroregeneration and neuroprotection. Besides, LGF stimulates the phosphorylation of MAPK/ERK1/2 and CREB, and regulates the expression of proteins which are critical for cell survival such as Bcl2 and Akt. Because LGF partially protects dopamine neurons from 6-OHDA neurotoxicity in the substantia nigra, and reduces motor deficits in these animals, we propose LGF as a novel factor that may be useful in the treatment of Parkinson's disease.
... 2 Laura E. O' Dell et al. Non-glycosylated DAT represents intracellular DAT levels, while glycosylated DAT represents cell surface DAT levels ( Afonso-Oramas et al. 2009). ...
Patients with diabetes display a heightened propensity to use tobacco; however, it is unclear whether they experience enhanced rewarding effects of nicotine. Thus, this study examined the reinforcing effects of nicotine in a rodent model of diabetes involving administration of streptozotocin (STZ), a drug that is toxic to pancreatic insulin-producing cells. The first study compared STZ- and vehicle-treated rats that had 23-hour access to intravenous self-administration (IVSA) of nicotine or saline and concomitant access to food and water. In order to examine the contribution of dopamine to our behavioral effects, dopamine transporter (DAT), D1 and D2 receptor levels were compared in the nucleus accumbens (NAc) following 10 days of nicotine or saline IVSA. Dopamine levels in the NAc were also compared following nicotine administration. Lastly, nicotine metabolism and dose-dependent effects of nicotine IVSA were assessed. The results revealed that STZ-treated rats displayed enhanced nicotine intake and a robust increase in food and water intake relative to controls. Protein analysis revealed an increase in DAT and a decrease in D1 receptor levels in the NAc of STZ- versus vehicle-treated rats regardless of IVSA condition. STZ-treated rats also displayed suppressed NAc dopamine levels during baseline and in response to nicotine. STZ treatment did not alter our assessment of nicotine metabolism. Furthermore, STZ treatment increased nicotine IVSA in a dose-dependent manner. Our findings suggest that STZ-treatment increased the rewarding effects of nicotine. This suggests that strong reinforcing effects of nicotine may contribute to greater tobacco use in patients with diabetes.
... Amongst other plausible hyperthermia-independent mechanisms underlying the neuroprotection observed after D2R inactivation, there is most consistent evidence for the fact that the activity of striatal DAT, which is crucial for methamphetamine-induced neurotoxicity on dopaminergic striatal terminals [50,150,151], is reduced in the absence of D2R [152]. This decrease in DAT activity produced by inactivation of the D2R seems to block dopamine reuptake, resulting in lower intracytosolic dopamine levels ( Fig. 4) which, as with inactivation of the D1R, are likely to mediate neuroprotection after D2R inactivation. ...
Methamphetamine is a synthetic drug consumed by millions of users despite its neurotoxic effects in the brain, leading to loss of dopaminergic fibres and cell bodies. Moreover, clinical reports suggest that methamphetamine abusers are predisposed to Parkinson's disease. Therefore, it is important to elucidate the mechanisms involved in methamphetamine-induced neurotoxicity. Dopamine receptors may be a plausible target to prevent this neurotoxicity. Genetic inactivation of dopamine D1 or D2 receptors protects against the loss of dopaminergic fibres in the striatum and loss of dopaminergic neurons in the substantia nigra. Protection by D1 receptor inactivation is due to blockade of hypothermia, reduced dopamine content and turnover and increased stored vesicular dopamine in D1R(-/-) mice. However, the neuroprotective impact of D2 receptor inactivation is partially dependent on an effect on body temperature, as well as on the blockade of dopamine reuptake by decreased dopamine transporter activity, which results in reduced intracytosolic dopamine levels in D2R(-/-) mice.
... The present data indicate that both DAT and VMAT2 expression in nonhuman primate striatum increases rapidly during the last half of gestation and that adult levels are achieved at the time of birth. Both transporters exist in glycosylated and native forms, and as the glycosylation status appears to affect the function of the proteins (Afonso Oramas et al., 2009;Cruz-Muros et al., 2008;Li et al., 2004), it is possible that an altered degree of glycosylation during development contributes to the observed change in transporter binding. ...
Several addictive or neurotoxic drugs are dependent on the dopamine transporter (DAT) and/or vesicular monoamine transporter (VMAT2) to exert their detrimental effects on dopamine neurons. For example, methamphetamine and MPTP are substrates for both DAT and VMAT2, with the ratio of DAT to VMAT2 in striatum being a determinant of the degree of toxicity inflicted by these drugs on dopamine neurons. Thus, the susceptibility of dopamine neurons to agents whose pharmacology involves DAT and VMAT2 may vary during development if the ontogeny of DAT and VMAT2 differ, and this is relevant as exposure of dopamine neurons to toxic agents during development is hypothesized to underlie some neurological or psychiatric disorders. However, the relative expression of DAT and VMAT2 has not been studied in either primate or non-primate fetal brain, and this was addressed in the present study by measuring the binding of specific radioligands of DAT and VMAT2 to striatal membranes from non-human primates at mid-gestation, late-gestation and the postnatal and adult periods. Dopamine concentration was also determined in striatal tissue from the same brains. These data indicate that in striatum of primates, unlike rodents, there is a sharp increase in DAT and VMAT2 expression after mid-gestation with adult levels being attained at the time of birth. In addition, this study demonstrated that there is a coordinated expression of DAT and VMAT2 from the time of mid-gestation to adulthood. Synapse, 2013. © 2013 Wiley Periodicals, Inc.
It is urgently needed to find reliable biofluid biomarkers for early diagnosis of Parkinson's disease in order to achieve better treatment. Promising biomarkers can be found in Parkinson's disease-related glycoproteins as aberrant protein glycosylation plays an important role in disease progression. However, current information on serum N-glycoproteomic changes in Parkinson's disease is still limited. Here, we used glycoproteomics methods, which combine the solid-phase chemoenzymatic method, lectin affinity chromatography, and hydrophilic interaction chromatography with high-resolution mass spectrometry, to analyze the glycans, glycosites, and intact glycopeptides of serum. Increased abundance of glycans containing core fucose, sialic acid, and bisecting N-acetyl glucosamine was detected at the overall glycan level and also at specific glycosites of glycopeptides. Five Parkinson's disease-associated proteins with this type of N-glycosylation changes were also identified. We propose that the revealed site-specific N-glycosylation changes in serum can be potential biomarkers for Parkinson's disease.
Co‐transmission of glutamate by brain dopaminergic neurons was recently proposed as a potential factor influencing cell survival in models of Parkinson’s disease. Intriguingly, brain dopaminergic nuclei are differentially affected in Parkinson’s disease. Whether this is associated with different patterns of co‐expression of the glutamatergic phenotype along the rostro‐caudal brain axis is unknown in mammals. We hypothesized that, as in zebrafish, the glutamatergic phenotype is present preferentially in the caudal meso‐diencephalic dopaminergic nuclei. Here, we used in mice a cell fate mapping strategy based on reporter protein expression (ZsGreen) consecutive to previous expression of the vesicular glutamate transporter 2 (Vglut2) gene, coupled with immunofluorescence experiments against tyrosine hydroxylase (TH) or dopamine transporter (DAT). We found three expression patterns in DA cells, organized along the rostro‐caudal brain axis. The first pattern (TH‐positive, DAT‐positive, ZsGreen‐positive) was found in A8‐A10. The second pattern (TH‐positive, DAT‐negative, ZsGreen‐positive) was found in A11. The third pattern (TH‐positive, DAT‐negative, ZsGreen‐negative) was found in A12‐A13. These patterns should help to refine the establishment of the homology of dopaminergic nuclei between vertebrate species. Our results also uncover that Vglut2 is expressed at some point during cell lifetime in dopaminergic nuclei known to degenerate in Parkinson's disease and largely absent from those that are preserved, suggesting that co‐expression of the glutamatergic phenotype in dopaminergic cells influences their survival in Parkinson’s disease.
Dutasteride is a 5alpha-reductase inhibitor in clinical use to treat endocrine conditions. The present study investigated the neuroprotective mechanisms of action of dutasteride in intact and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice using a low dose of MPTP not affecting motor activity modeling early stages of Parkinson's disease (PD). We hypothesized that dutasteride neuroprotection is due to altered steroids levels. Dutasteride pre-treatment prevented loss of striatal dopamine (DA) and its metabolite DOPAC. Dutasteride decreased effects of MPTP on striatal dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2) and D2 DA receptor specific binding while D1 receptor specific binding remained unchanged. Dutasteride enhanced DAT specific binding and the glycosylated form of DAT in intact mice. MPTP-lesioned mice had plasma and brain testosterone and dihydrotestosterone levels lower than control mice whereas progesterone and its metabolites (dihydroprogesterone, isopregnanolone and tetrahydroprogesterone) pathway showed increases. Dutasteride treatment by inhibiting transformation of progesterone and testosterone to its metabolites elevated plasma and brain concentrations of testosterone compared to MPTP mice and decreased DHT levels in intact mice. Plasma and brain estradiol levels were low and remained unchanged by MPTP and/or dutasteride treatment. Dutasteride treatment did not affect striatal phosphorylation of Akt and its downstream substrate GSK3β as well as phosphorylation of ERK1/2 in intact and MPTP lesioned MPTP mice. Striatal glial fibrillary acidic protein (GFAP) levels were markedly elevated in MPTP compared to control mice and dutasteride reduced GFAP levels in MPTP mice. Treatment with dutasteride post-lesion left unchanged striatal DA levels. These results suggest dutasteride as promising drug for PD neuroprotection.
Many clinical studies have reported on the benefits of exercise therapy in patients with Parkinson's disease (PD). Exercise cannot stop the progression of PD or facilitate the recovery of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (Bega et al., 2014). To tease apart this paradox, we utilized a progressive MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetra-hydropyridine) mouse model in which we initiated 4 weeks of treadmill exercise after the completion of toxin administration (i.e., restoration). We found in our MPTP/exercise (MPTP+EX) group several measures of gait function that recovered compared to the MPTP only group. Although there was a small recovery of tyrosine hydroxylase (TH) positive DA neurons in the SNpc and terminals in the striatum, this increase was not statistically significant. These small changes in TH could not explain the improvement of motor function. The MPTP group had a significant 170% increase in the glycosylated/non-glycosylated dopamine transporter (DAT) and a 200% increase in microglial marker, IBA-1, in the striatum. The MPTP+EX group showed a near full recovery of these markers back to the vehicle levels. There was an increase in GLT-1 levels in the striatum due to exercise, with no change in striatal BDNF protein expression. Our data suggests that motor recovery was not prompted by any significant restoration of DA neurons or terminals, but rather the recovery of DAT and dampening the inflammatory response. Although exercise does not promote recovery of nigrostriatal DA, it should be used in conjunction with pharmaceutical methods for controlling PD symptoms.
Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc⁻, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc⁻. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6 J mice receiving repeated intraperitoneal injections of either ZNS 30 mg kg− 1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post-surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc⁻ was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6 J mice, nor did it alter system xc⁻ activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc⁻ independent mechanisms.
Amphetamine-related drugs, such as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH), are popular recreational psychostimulants. Several preclinical studies have demonstrated that, besides having the potential for abuse, amphetamine-related drugs may also elicit neurotoxic and neuroinflammatory effects. The neurotoxic potentials of MDMA and METH to dopaminergic and serotonergic neurons have been clearly demonstrated in both rodents and non-human primates. This review summarizes the species-specific cellular and molecular mechanisms involved in MDMA and METH-mediated neurotoxic and neuroinflammatory effects, along with the most important behavioral changes elicited by these substances in experimental animals and humans. Emphasis is placed on the neuropsychological and neurological consequences associated with the neuronal damage. Moreover, we point out the gap in our knowledge and the need for developing appropriate therapeutic strategies to manage the neurological problems associated with amphetamine-related drug abuse.
Liver growth factor (LGF) is a hepatic mitogen which has the ability to stimulate tissue regeneration and cell proliferation in both liver and extrahepatic tissues. The aim of this work was to evaluate the regenerative and / or neuroprotective effects, and the stimulation of foetal and adult neural stem cells (NSC), exerted by LGF treatment in a rat model of Parkinson's disease (PD) induced by 6-hidroxydopamine (6-OHDA).
The study of the effects carried out by LGF administration showed that this factor stimulates neurogenesis and cell survival, promotes migration of newly generated neurons, and induces the outgrowth of tyrosine hydroxylase-positive terminals in the lesioned striatum. Furthermore, LGF treatment raises striatal dopamine levels and partially protects dopaminergic neurons of the substantia nigra of hemiparkinsonian animals from 6-OHDA neurotoxicity. LGF also stimulates survival of grafted foetal NSC´s in the dopamin depleted striatum, reduces apomorphine-induced rotational behaviour and improves motor coordination in this experimental model of PD. The results indicate that microglia could be the cellular target of LGF in the central nervous system. Finally, the activity of LGF could be mediated by the stimulation of MAPK/ERK1/2 signalling pathway and by regulating critical proteins for cell survival, such as Bcl-2 and phospho-CREB.
Since LGF showed neuroprotective, neuroregenerative and neurogenic effects in 6-OHDA-lesioned rats, we propose LGF as a novel factor that may be useful in the treatment of Parkinson’s disease.
Numerous G protein-coupled receptors (GPCRs) are glycosylated at extracellular regions. The regulatory roles of glycosylation on receptor function vary across receptor types. In this study, we used the dopamine D2 and D3 receptors as an experimental model to understand the underlying principles governing the functional roles of glycosylation. We used the pharmacological inhibitor, tunicamycin, to inhibit glycosylation, generated chimeric D2 and D3 receptors by swapping their respective N-termini, and produced the glycosylation site mutant D2 and D3 receptors to study the roles of glycosylation on receptor functions, including cell surface expression, signaling, and internalization through specific microdomains. Our results demonstrate that glycosylation on the N-terminus of the D3 receptor is involved in the development of desensitization and proper cell surface expression. In addition, glycosylation on the N-terminus mediates the internalization of D2 and D3 receptors within the caveolae and clathrin-coated pit microdomains of the plasma membrane, respectively, by regulating receptor interactions with caveolin-1 and clathrin. In conclusion, this study shows for the first time that glycosylation on the N-terminus of GPCRs is involved in endocytic pathway selection through specific microdomains. These data suggest that changes in the cellular environment that influence posttranslational modification could be an important determinant of intracellular GPCR trafficking.
On the basis of behavioural evidence, dopamine is found to be involved in two higher-level functions of the brain: reward-mediated learning and motor activation. In these functions dopamine appears to mediate synaptic enhancement in the corticostriatal pathway. However, in electrophysiological studies, dopamine is often reported to inhibit corticostriatal transmission. These two effects of dopamine seem incompatible. The existence of separate populations of dopamine receptors, differentially modulating cholinergic and glutamatergic synapses, suggests a possible resolution to this paradox.
The synaptic enhancement which occurs in reward-mediated learning may also be involved in dopamine-mediated motor activation. The logical form of reward-mediated learning imposes constraints on which mechanisms can be considered possible. Dopamine D1 receptors may mediate enhancement of corticostriatal synapses. On the other hand, dopamine D2 receptors on cholinergic terminals may mediate indirect, inhibitory effects of dopamine on striatal neurons.
The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, 125I-1-[2-(diphenylmethoxy)ethyl]-4-[2- (4-azido-3-iodophenyl)ethyl]piperazine (125I-DEEP). 125I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, 125I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.
A syndrome similar to idiopathic parkinsonism developed after intravenous self-administration of an illicit drug preparation in which N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP) might have been responsible for the toxicity. In the present study we show that intravenous administration of NMPTP to the rhesus monkey produces a disorder like parkinsonism (akinesia, rigidity, postural tremor, flexed posture, eyelid closure, drooling) that is reversed by the administration of L-dopa. NMPTP treatment decreases the release of dopamine and dopamine accumulates in swollen, distorted axons in the nigrostriatal pathway just above the substantia nigra, followed by severe nerve cell loss in the pars compacta of the substantia nigra and a marked reduction in the dopamine content of the striatum. The pathological and biochemical changes produced by NMPTP are similar to the well-established changes in patients with parkinsonism. Thus, the NMPTP-treated monkey provides a model that can be used to examine mechanisms and explore therapies of parkinsonism.
Synapsin I (protein I) is a neuron-specific phosphoprotein, which is a substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases. In two accompanying studies (De Camilli, P., R. Cameron, and P. Greengard, and De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354 and 1355-1373) we have shown, by immunocytochemical techniques at the light microscopic and electron microscopic levels, that synapsin I is present in the majority of, and possibly in all, nerve terminals, where it is primarily associated with synaptic vesicles. In the present study we have prepared a highly purified synaptic vesicle fraction from rat brain by a procedure that involves permeation chromatography on controlled-pore glass as a final purification step. Using immunological methods, synapsin I concentrations were determined in various subcellular fractions obtained in the course of vesicle purification. Synapsin I was found to copurify with synaptic vesicles and to represent approximately 6% of the total protein in the highly purified synaptic vesicle fraction. The copurification of synapsin I with synaptic vesicles was dependent on the use of low ionic strength media throughout the purification. Synapsin I was released into the soluble phase by increased ionic strength at neutral pH, but not by nonionic detergents. The highly purified synaptic vesicle fraction contained a calcium-dependent protein kinase that phosphorylated endogenous synapsin I in its collagenase-sensitive tail region. The phosphorylation of this region appeared to facilitate the dissociation of synapsin I from synaptic vesicles under the experimental conditions used.
Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C-terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.
In urethane-anesthetized rats, a membrane potential oscillation (MPO) of up to 30 mV and 0.5-2 Hz (delta frequency range) was found in neurons of the nucleus accumbens. The membrane potential oscillations were of similar frequency and reversed in phase to the extracellular EEG of about 0.5 mV. In freely moving rats, a rhythmic delta EEG of 0.5-3 Hz was found in the nucleus accumbens, and it was of highest amplitude and regularity during awake immobility and face washing, less regular during slow wave sleep, and of the lowest amplitude during body and head movements and rapid eye movement sleep. The behavioural relation of the accumbens EEG was not critically affected after amphetamine, haloperidol, and parachlorophenylalanine, which depleted serotonin, although the accumbens EEG during awake immobility was less regular after blocking muscarinic cholinergic receptors by atropine sulfate. However, stimulation of the ventral tegmental area suppressed the accumbens delta membrane potential oscillations and EEG, and this effect was antagonized by haloperidol, suggesting that endogenous dopamine release may suppress the accumbens delta rhythm. It was concluded that the delta rhythm in the nucleus accumbens may represent a state of bilateral synchrony among accumbens neurons that is perhaps characteristic of an idling system, while desynchronization of the delta rhythm may occur closely with motor action.
The uptake of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was studied in various mammalian cell lines transfected, respectively, with the cloned human and rat dopamine transporters, and compared with rat striatal synaptosome preparations. Only in neuronally derived cell lines such as NG108-15, NS20Y, and SK-N-MC cells did MPP+ have a KM for the cloned transporters comparable to that of dopamine as seen in rat striatal synaptosomes. In non-neuronally derived cells such as COS-7, CHO, and Ltk- cells transiently or permanently expressing the transporters, the KM of MPP+ was at least 10-fold higher. The permanent expression of either the cloned human or rat dopamine transporters conferred to SK-N-MC cells susceptibility to the cytotoxic effects of low concentrations of MPP+. The extent of this effect was dependent on the expression level of the dopamine transporters and could be specifically antagonized by the catecholamine uptake inhibitor mazindol. There were no significant differences in the susceptibility to MPP+ of cells expressing similar levels of either the human or rat dopamine transporter. The demonstration for the first time of a quantitative relationship between the cellular expression of the plasma membrane transporter and the extent of the cytotoxic effects of MPP+ suggests that known differences in vulnerability of various brain regions to MPP+ cytotoxicity might be related to their actual content of dopamine uptake sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Purpose of review In the past 18 months, several important studies on the epidemiology of Parkinson's disease have been published. In particular, large cohorts have identified sufficient incident patients with Parkinson's disease to study risk or protective factors of Parkinson's disease; one of the important recent events in the field is the publication of some of their findings. Recent findings We will first review findings of descriptive studies on the frequency of the disease and its geographic or temporal distribution. We will then summarize the findings of analytical studies dealing with risk or protective factors in the fields of dietary and lifestyle factors (cigarette smoking, coffee and tea drinking, uric acid, dairy products), environmental exposures (pesticides, lead, manganese, welding), hormonal factors (oophorectomy), vascular risk factors (diabetes, hypertension, cholesterol level), pharmacoepidemiology (NSAIDs, statins), and familial aggregation. Epidemiologic studies have consistently found that some exposures are inversely (e.g., cigarette smoking) or positively associated with Parkinson's disease (e.g., pesticides), while their findings are, at the present time, less consistent for other exposures (e.g., NSAIDs, vascular risk factors). Finally, recent studies have investigated new research fields (e.g., hormonal factors, uric acid, pharmacoepidemiology) and additional data need to be collected.
Parkinson's disease is characterized by massive degeneration of dopamine-containing neurons in the midbrain. However, the vulnerability of these neurons is heterogeneous both across different midbrain dopamine-containing cell groups and within the substantia nigra, the brain structure most affected in this disease. To determine the exact pattern of cell loss and to map the cellular distribution of candidate pathogenic molecules, it is necessary to have landmarks independent of the degenerative process by which to subdivide the substantia nigra. We have developed a protocol for this purpose based on immunostaining for calbindin D28K, a protein present in striatonigral afferent fibres. We used it to examine post-mortem brain samples from seven subjects who had had no history of neurological or psychiatric disease. We found intense immunostaining for calbindin D28K associated with the neuropil of the ventral midbrain. Within the calbindin-positive region, there were conspicuous calbindin-poor zones. Analysed in serial sections, many of the calbindin-poor zones seen in individual sections were continuous with one another, forming elements of larger, branched three-dimensional structures. Sixty per cent of all dopamine-containing neurons in the substantia nigra pars compacta were located within the calbindin-rich zone, which we named the nigral matrix, and 40% were packed together within the calbindin-poor zones, which we named nigrosomes. We identified five different nigrosomes. This organization was consistent from one control brain to another. We propose that subdivision of the human substantia nigra based on patterns of calbindin immunostaining provides a key tool for analysing the organization of the substantia nigra and offers a new approach to analysing molecular expression patterns in the substantia nigra and the specific patterns of nigral cell degeneration in Parkinson's disease.
To achieve accuracy in studying the patterns of loss of midbrain dopamine-containing neurons in Parkinson's disease, we used compartmental patterns of calbindin D 28K immunostaining to subdivide the substantia nigra with landmarks independent of the degenerative process. Within the substantia nigra pars compacta, we identified dopamine-containing neurons in the calbindin-rich regions ('matrix') and in five calbindin-poor pockets ('nigrosomes') defined by analysis of the three-dimensional networks formed by the calbindin-poor zones. These zones were recognizable in all of the brains, despite severe loss of dopamine-containing neurons. The degree of loss of dopamine-containing neurons in the substantia nigra pars compacta was related to the duration of the disease, and the cell loss followed a strict order. The degree of neuronal loss was significantly higher in the nigrosomes than in the matrix. Depletion was maximum (98%) in the main pocket (nigrosome 1), located in the caudal and mediolateral part of the substantia nigra pars compacta. Progressively less cell loss was detectable in more medial and more rostral nigrosomes, following the stereotyped order of nigrosome 1 > nigrosome 2 > nigrosome 4 > nigrosome 3 > nigrosome 5. A parallel, but lesser, caudorostral gradient of cell loss was observed for dopamine-containing neurons included in the matrix. This pattern of neuronal loss was consistent from one parkinsonian substantia nigra pars compacta to another. The spatiotemporal progression of neuronal loss related to disease duration can thus be drawn in the substantia nigra pars compacta for each Parkinson's disease patient: depletion begins in the main pocket (nigrosome 1) and then spreads to other nigrosomes and the matrix along rostral, medial and dorsal axes of progression.
Parkinson's disease occurs in 1percent of people over the age of 65 when about 60percent of the dopaminergic neurons in the substantia nigra of the midbrain are lost. Dopaminergic neurons appear to die by a process of apoptosis that is induced by oxidative stress. Oxygen radicals abstract hydrogen from DNA forming DNA radicals that lead to DNA fragmentation, activation of DNA protective mechanisms, NAD depletion and apoptosis. Oxygen radicals can be formed in dopaminergic neurons by redox cycling of MPP+ , the active metabolite of MPTP. This redox cycling mechanism involves the reduction of MPP+ by a number of enzymes, especially flavin containing enzymes, some of which are found in mitochondria. Tyrosine hydroxylase is present in all dopaminergic neurons and is responsible for the synthesis of dopamine. However, tyrosine hydroxylase can form oxygen radicals in a redox mechanism involving its cofactor, tetrahydrobiopterin. Dopamine may be oxidized by monoamine oxidase to form oxygen radicals and 3,4-dihydroxyphenylacetaldehyde. This aldehyde may be oxidized by aldehyde dehydrogenase with the formation of oxygen radicals and 3,4-dihydroxyphenylacetic acid. The redox mechanisms of oxygen radical formation by MPTP, tyrosine hydroxylase, monoamine oxidase and aldehyde dehydrogenase will be discussed. Possible clinical applications of these mechanisms will be briefly presented.
Active uptake of 3,4-dihydroxyphenylethylamine (dopamine) is sodium- and temperature-dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+-dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low- and high-affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.
The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29-fold increase in the level of GFAP mRNA in the striatum of wild-type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+; 10 mM), via a microdialysis probe produced a massive efflux of DA in wild-type mice (>320-fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.
Regional blood flow distribution in ducks was estimated from fractional distribution of Rb86CI. A comparison was made between normally breathing ducks and submerged ducks. The data document that conspicuous changes in regional blood flow take place upon submersion. The skin, skeletal muscle, and organs of the gastrointestinal system showed a marked decrease in activity in the submerged condition down to an average of 3.9 % for the gizzard, 29.4 % for skin excised from the thorax and 11.0 % for gastrocnemius muscle. Surprising exceptions to this were displayed by tissues in the cranial part of the animals. Thus both skin and muscle from the head region showed an increase in activity during submersion. The same was true for the excised eye. The esophagus similarly showed an increased activity in the submerged animals. The myocardium from both atria and ventricles showed a striking increase amounting to 4.1 times higher activity in the left ventricular myocardium in the submerged animals. The change in vasomotor constrictor tone taking place upon submersion is thus highly selective and possibly segmentally oriented giving an increase in peripheral resistance to most organs posterior to the heart while the most cranial tissues seem to be subjected to a general decrease in vasoconstrictor tone with an increased blood flow. Activity in the kidneys was markedly consistent and 9.1 % of the value found in normally breathing animals. The adrenals showed an interesting increase in activity on the average as high as 4.9 times the value in normally breathing animals.
Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for cross-reactivity to four epitope-specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of ∼80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N-linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms <80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti-rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.
The forebrain is characterized by a dense, localized dopamine (DA) innervation pattern, a diffuse, widespread norepinephrine (NE) innervation pattern, and a serotonin (5-HT) innervation intermediate between the DA and NE patterns. These innervation patterns have implied that basic differences exist in the way DA, NE and 5-HT axons collateralize to different brain structures; that is, DA axons are thought to be poorly collateralized and NE and 5-HT axons are presumed to be more highly collateralized. In the present study, we used injections of retrograde labeling fluorescent dyes into various forebrain regions in order to determine axonal branching patterns from nuclei that contain DA, NE and 5-HT neurons, namely the substantia nigra-ventral tegmental area (SN-VTA), locus coeruleus (LC) and raphe nuclei (DR-MR). The results suggest that at least two subpopulations of neurons can be defined in each monoamine nucleus with respect to the way their axons collateralize. Each area contains a centrally located nuclear area with highly collateralized neurons, and more peripherally situated areas with less highly collateralized neurons. Thus, previous suppositions of the branching of monoamine axons must be revised to account for the existence of cells exhibiting totally different Collateralization patterns within each monoamine nucleus.
Fast cyclic voltammetry was used in the caudate and nucleus accumbens of anaesthetised rats to study the release and reuptake of dopamine following stimulation of the median forebrain bundle. Dopamine uptake was significantly slower in accumbens than caudate, indicating a lower number of functional uptake sites. This implies that dopamine may be able to diffuse further from its sites of release in nucleus accumbens than in caudate and thus may have a neuromodulator role in the region.
Braak and colleagues have proposed that, within the central nervous system, Parkinson's disease (PD) begins as a synucleinopathy in nondopaminergic structures of the lower brainstem or in the olfactory bulb. The brainstem synucleinopathy is postulated to progress rostrally to affect the substantia nigra and cause parkinsonism at a later stage of the disease. In the context of a diagnosis of PD, made from current clinical criteria, the pattern of lower brainstem involvement accompanying mesencephalic synucleinopathy is often observed. However, outside of that context, the patterns of synucleinopathy that Braak described are often not observed, particularly in dementia with Lewy bodies and when synucleinopathy occurs in the absence of neurological manifestations. The concept that lower brainstem synucleinopathy represents "early PD" rests on the supposition that it has a substantial likelihood of progressing within the human lifetime to involve the mesencephalon, and thereby cause the substantia nigra pathology and clinical parkinsonism that have heretofore defined the disease. However, the predictive validity of this concept is doubtful, based on numerous observations made in populations of aged individuals who, despite the absence of neurological signs, have brain synucleinopathy ranging up to Braak stages 4 to 6 at postmortem. Furthermore, there is no relation between Braak stage and the clinical severity of PD. We conclude that the relation between patterns of abnormal synuclein immunostaining in the human brain and the disease entity now recognized as PD remains to be determined.
Computer imaging and immunohistochemical staining techniques were used to determine which midbrain dopaminergic (DA) cells are spared in Parkinson's disease (PD), and in animals treated with the DA neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and whether the spared cells contain the calcium-binding protein, calbindin-D28k (CaBP). The PD patients had more than 55% fewer midbrain DA neurons than age-matched normal subjects. The cell loss occurred within the combined substantia nigra and retrorubral area (greater than 61%; DA nuclei A9 and A8, respectively), and the ventral tegmental area (greater than 42%; DA nucleus A10). The cell loss was greatest within the ventral portion of the nucleus A9. A similar pattern of DA cell loss was observed in MPTP-treated Macaca fascicularis monkeys. The CaBP-containing cells were located specifically in the cell regions spared by PD and by MPTP-treatment in both monkeys and C57BL/6 mice. These data suggest that PD and MPTP both destroy the same population of midbrain DA neurons within nuclei A8, A9, and A10, and that perhaps CaBP protects the DA neurons from cell death caused by both PD and MPTP.
Oligonucleotides and a full-length cDNA encoding a functional dopamine transporter (DAT1) hybridize to a 3.7 kb mRNA that is concentrated in mRNA prepared from midbrain and absent in specimens from cerebellum or cerebral cortex. In situ hybridization reveals substantial hybridization densities overlying neurons of the substantia nigra, pars compacta, and the parabrachialis pigmentosus region of the ventral tegmental area (VTA). Neurons in the linear and paranigral VTA regions display lower levels of expression. Preliminary studies in arcuate neurons suggest modest hybridization. Different dopaminergic cell groups display different levels of DAT1 dopamine transporter expression.
Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCl (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopamine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine.
The dopamine transporter from rat caudate-putamen was photolabeled with [125I]DEEP as previously described. Treatment of photolabeled membranes with neuraminidase and N-glycanase reduced the molecular weight of the [125I]DEEP photolabeled dopamine transporter complex, whereas treatment with alpha-mannosidase had no effect. The solubilized [125I]DEEP photolabeled dopamine transporter complex readily bound to wheat-germ agglutinin but not to concanavalin-A sepharose columns. These results suggest that the carbohydrate moiety of the dopamine transporter is N-linked and contains significant quantities of sialic acid but not high mannose residues. A DEEP binding protein was readily detectable in other brain regions including the nucleus accumbens and olfactory tubercle, but not in the prefrontal cortex, olfactory bulb or hypothalamus under similar conditions. The DEEP binding protein in the other brain regions was similar to that in the striatum.
Quantitative measures for the severity of MPTP-induced parkinsonism and response to antiparkinsonian interventions in monkeys have been lacking. We carried out an oral levodopa dose-response study in two rhesus monkeys whose left hemiparkinsonism was induced by intracarotid administration of MPTP. A newly developed clinical rating scale of monkey parkinsonism showed a consistent dose-response relationship for levodopa over the dosage range of 50-3,500 mg/day. Antiparkinsonian effects appeared at 200 mg/day and were optimal at 1,000-2,000 mg/day. Levodopa also reversed rotational behavior, improved movement times for both the impaired and opposite upper limb, and produced dyskinesias at high dosages. Thus, MPTP-induced hemiparkinsonism in monkeys closely resembles the human disease condition, is associated with sensitive response measures, and should prove valuable for assessing novel antiparkinsonian therapies.
Using positron emission tomography (PET), we studied regional striatal 18F-dopa uptake in 16 patients with L-dopa-responsive Parkinson's disease (PD), 18 patients with multiple system atrophy, and 10 patients with progressive supranuclear palsy. Results were compared with those of 30 age-matched normal volunteers. The patients with PD showed significantly reduced mean uptake of 18F-dopa in the caudate and putamen compared to controls, but while function in the posterior part of the putamen was severely impaired (45% of normal), function in the anterior part of the putamen and in the caudate was relatively spared (62% and 84% of normal). Mean 18F-dopa uptake in the posterior putamen was depressed to similar levels in all patients. Unlike patients with PD, the patients with progressive supranuclear palsy showed equally severe impairment of mean 18F-dopa uptake in the anterior and posterior putamen. Caudate 18F-dopa uptake was also significantly lower in patients with progressive supranuclear palsy than in patients with PD, being depressed to the same level as that in the putamen. Mean 18F-dopa uptake values in the anterior putamen and caudate in patients with multiple system atrophy lay between PD and progressive supranuclear palsy levels. Locomotor disability of individual patients with PD or multiple system atrophy correlated with decline in striatal 18F-dopa uptake, but this was not the case for the patients with progressive supranuclear palsy. We conclude that patients with PD have selective nigral pathological features with relative preservation of the dopaminergic function in the anterior putamen and caudate, whereas there is progressively more extensive nigral involvement in multiple system atrophy and progressive supranuclear palsy.(ABSTRACT TRUNCATED AT 250 WORDS)
Investigations were conducted to determine the topography of the high-affinity dopamine uptake process within the rat striatum. [³H]Dopamine uptake into crude synaptosomes prepared from micropunch samples was found to be two- to three-fold higher in dorsal caudate-putamen relative to nucleus accumbens septi. In contrast, the concentrations of dopamine in the two regions were equivalent. The recognition site associated with high-affinity dopamine uptake was labeled using [³H]mazindol, and the binding of this ligand was also found to be two- to three-fold higher in homogenates from dorsal caudate-putamen samples relative to nucleus accumbens septi. Regional differences in uptake of [³H]dopamine or binding of [³H]mazindol were shown to be due to variations in Vmax or Bmax, not to differences in apparent affinity. Autoradiography of [³H]mazindol binding in rat striatum revealed a decreasing density of the site along the dorsal-to-ventral axis, with the highest binding occurring in the dorsolateral caudate-putamen, lower binding in the ventral caudate-putamen, and lowest levels in the septal pole of the nucleus accumbens septi. Quantification showed that the extent of this gradient was two-fold. Further autoradiographic studies revealed less striatal heterogeneity in the pattern of binding of [³H]ketanserin, another radioligand associated with the striatal dopaminergic innervation but not linked to the dopamine uptake process of the plasma membrane.
The distribution of calbindin-D28K (CaBP)-positive neurons was investigated by immunohistochemistry in 4 controls, 5 cases of Parkinson's disease and a single case of strionigral degeneration. CaBP-positive neurons were preferentially localized to the mediodorsal portion of the substantia nigra pars compacta (SNC) in the beta layer, while CaBP-negative, melanin-positive neurons were concentrated in the ventrolateral SNC in the alpha layer. In Parkinson's disease and the case of strionigral degeneration, there was a relative sparing of the CaBP-positive neurons compared with CaBP-negative, pigmented neurons. These data imply that CaBP may confer some protection to SNC dopaminergic neurons against the pathological process which is responsible for Parkinson's disease and strionigral degeneration.
Computer visualization techniques were used to map the distribution of dopaminergic neurons within midbrain tissue sections from 5 parkinsonian patients and 3 age-matched control subjects. The Parkinsonian brains had over 50% fewer dopaminergic neurons within the midbrain than age-matched normal brains. The cell loss occurred within the combined substantia nigra (dopaminergic nucleus A9) and retrorubral (dopaminergic nucleus A8) areas (greater than 61%) and the ventral tegmental area (dopaminergic nucleus A10) (greater than 42%). The cell loss was greatest within the ventral portion of the substantia nigra zona compacta. The specific pattern of cell loss is very similar to the pattern of cells that project to the striatum (as opposed to cortical and limbic sites) in animal neuroanatomical tracing experiments. These data suggest that Parkinson's disease preferentially destroys midbrain dopaminergic neurons in nuclei A8, A9, and A10, which project to the striatum.
In idiopathic Parkinson's disease massive cell death occurs in the dopamine-containing substantia nigra. A link between the vulnerability of nigral neurons and the prominent pigmentation of the substantia nigra, though long suspected, has not been proved. This possibility is supported by evidence that N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite MPP+, the latter of which causes destruction of nigral neurons, bind to neuromelanin. We have directly tested this hypothesis by a quantitative analysis of neuromelanin-pigmented neurons in control and parkinsonian midbrains. The findings demonstrate first that the dopamine-containing cell groups of the normal human midbrain differ markedly from each other in the percentage of neuromelanin-pigmented neurons they contain. Second, the estimated cell loss in these cell groups in Parkinson's disease is directly correlated (r = 0.97, P = 0.0057) with the percentage of neuromelanin-pigmented neurons normally present in them. Third, within each cell group in the Parkinson's brains, there is greater relative sparing of non-pigmented than of neuromelanin-pigmented neurons. This evidence suggests a selective vulnerability of the neuromelanin-pigmented subpopulation of dopamine-containing mesencephalic neurons in Parkinson's disease.
Autografting of dopamine-producing adrenal medullary tissue to the striatal region of the brain is now being attempted in patients with Parkinson's disease. Since the success of this neurosurgical approach to dopamine-replacement therapy may depend on the selection of the most appropriate subregion of the striatum for implantation, we examined the pattern and degree of dopamine loss in striatum obtained at autopsy from eight patients with idiopathic Parkinson's disease. We found that in the putamen there was a nearly complete depletion of dopamine in all subdivisions, with the greatest reduction in the caudal portions (less than 1 percent of the dopamine remaining). In the caudate nucleus, the only subdivision with severe dopamine reduction was the most dorsal rostral part (4 percent of the dopamine remaining); the other subdivisions still had substantial levels of dopamine (up to approximately 40 percent of control levels). We propose that the motor deficits that are a constant and characteristic feature of idiopathic Parkinson's disease are for the most part a consequence of dopamine loss in the putamen, and that the dopamine-related caudate deficits (in "higher" cognitive functions) are, if present, less marked or restricted to discrete functions only. We conclude that the putamen--particularly its caudal portions--may be the most appropriate site for intrastriatal application of dopamine-producing autografts in patients with idiopathic Parkinson's disease.
The high-affinity, sodium-dependent uptake of dopamine (DA) was inhibited by the pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. The inhibition was of a non-competitive type, resulting in a 40% decrease of Vmax. Neither basal nor depolarization-stimulated release of DA was affected. Treatment of synaptosomes with neuraminidase caused a 48% loss of sialic acid from the lipid-bound pool and a 80% decrease in the protein-bound fraction. The inhibition of DA uptake was found to be related linearly to the loss of sialic acid from the protein pool. It is postulated that a sialic acid moiety is involved in DA transport across the synaptosomal membrane.
A clinical, morphological and neurochemical correlative study in patients with Parkinson's syndrome and Huntington's chorea is reported. In the former group of patients, 69 brains were examined morphologically and 28 biochemically; in the latter group, 4 brains were examined morphologically and 14 biochemically. The results were as follows: (1) The main morphological alteration common to all forms of Parkinsonism was damage to the substantia nigra with a loss of the melanin-containing nerve cells in the zona compacta; the degree and pattern of this cell loss varied in a manner characteristic of the different forms of Parkinsonism. (2) Neurochemically, Parkinson's syndrome was characterized by a decrease in the concentrations of dopamine (DA) and its metabolite homovanillic acid (HVA) in the striatum (caudate nucleus and putamen) and pallidum. (3) A satisfactory positive correlation could be established between the degree of cell loss in the zona compacta of the substantia nigra and the disturbance of DA metabolism in the nuclei of the basal ganglia. (4) The nosological classification of Parkinson's syndrome (based on clinical and morphological criteria) into 3 main groups, viz. postencephalitic, idiopathic and “arteriosclerotic”-senile, was supported by distinct, though not in all cases statistically significant, differences in the degree of disturbance of striatal DA metabolism. (5) In general, mild Parkinsonism, just manifest clinically, was associated with a disproportionately high degree of striatal DA deficiency. From this it was concluded that: (a) clinically manifest Parkinsonism represents the late, “decompensated”, stage of a disease characterized by a progressive striatal DA deficiency; and (b) the striatum can compensate functionally for lower degrees of DA deficiency. (6) Positive correlations could be established, within a certain range, between the severity of individual Parkinsonian symptoms (especially akinesia and tremor) and (a) the degree, and also the site, of the disturbance of DA metabolism within the nuclei of the basal ganglia; and (b) the sensitivity of the patients to levodopa's acute anti-akinesia effect. The latter observation was interpreted as suggesting that in Parkinsonism there exists a supersensitivity (probably of the “denervation type”) of striatal receptors to DA. (7) Based on the correlations, levodopa therapy can be regarded as a specific, though probably predominantly symptomatic, treatment of the main extrapyramidal symptoms of Parkinson's syndrome. (8) In Huntington's chorea the concentrations of DA and HVA in the putamen, pallidum and substantia nigra showed no significant deviation from control values; however in the caudate nucleus there was a small but statistically significant reduction in the levels of these compounds (9). The possible functional significance of the observed correlations for the pathophysiology of disorders of the basal ganglia such as the diseases of Parkinson and Huntington is discussed.
The dopamine transporter (DAT) is a primary site for the action of cocaine in inducing euphoria. Its action is necessary for the selectivities of dopaminergic neurotoxins that provide the best current experimental models of Parkinson's disease. In the present report, rat dopamine transporter-like immunoreactivity (iDAT) was assessed by immunohistochemistry using newly developed polyclonal antisera raised against conjugated peptides corresponding to sequences found in the dopamine transporter's carboxy- and amino-termini. Dense iDAT was observed in patterns consistent with neural processes and terminals in the striatum, nucleus accumbens, olfactory tubercle, nigrostriatal bundle, and lateral habenula. Perikarya in the substantia nigra pars compacta were immunostained with moderate intensity using one of two immunohistochemical methods, while scattered ventral tegmental area perikarya were stained with somewhat less intensity. Immunoreactive neuronal processes with axonal and dendritic morphologies were stained in the substantia nigra and the paranigral and parabrachialis pigmentosus nuclei of the ventral tegmental area, while sparser processes were noted more medially in the ventral tegmental area. Neuronal processes were found in several laminae in the cingulate cortex, with notable fiber densities in the superficial aspects of lamina I and laminae II/III. The intensities of immunoreactivities in striatum and cerebral cortex were dramatically attenuated ipsilateral to nigrostriatal bundle 6-hydroxydopamine lesions. Specificity of immunostaining was supported by agreement of the results using sera directed against two distinct DAT segments, studies with preimmune and preadsorbed sera and studies of the extracted protein. These antisera identify and reveal details of the distribution of DAT immunoreactivity in rat brain and display variations in levels of DAT expression of likely functional significance.
The release of dopamine in the striatum, nucleus accumbens, and olfactory tubercle of anesthetized rats was evoked by electrical stimulation of the mesolimbic dopaminergic pathway (four pulses at 15 Hz or four pulses at 200 Hz). Carbon fiber electrodes were implanted in these regions to monitor evoked dopamine overflow by continuous amperometry. The kinetics of dopamine elimination were estimated by measuring the time to 50% decay of the dopamine oxidation current after stimulation ceased. This time ranged from 64 ms in the striatum to 113 ms in the nucleus accumbens. Inhibition of dopamine uptake by nomifensine (2-20 mg/kg), GBR 12909 (20 mg/kg), cocaine (20 mg/kg), mazindol (10 mg/kg), or bupropion (25 mg/kg) enhanced this decay time by up to +602%. Uptake inhibition also produced an increase in the maximal amplitude of dopamine overflow evoked by four pulses at 15 Hz. This latter effect was larger in the striatum (+420%) than in mesolimbic areas (+140%). These results show in vivo that these uptake inhibitors actually slow the clearance of dopamine released by action potentials and suggest that dopaminergic transmission is both prolonged and potentiated strongly by these drugs, in particular in the striatum.
Binding domains on rat dopamine transporters for cocaine and 1-(2-diphenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine compounds were identified using controlled proteolysis of photoaffinity-labeled protein and epitope-specific immunoprecipitation of the labeled fragments. Rat dopamine transporters were photoaffinity labeled with 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido- 3-[125I]iodophenyl)ethyl]piperazine ([125I]DEEP) [a 1-(2-di- phenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine analog] or 3 beta-(p-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'- [125I]iodophenylethyl ester ([125I]RTI 82) (a cocaine analog) and were gel purified to remove contaminating radioactivity. The resulting samples were treated with V8 protease or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide maps generated with each enzyme were different for each of the ligands, suggesting that the ligands were incorporated into different regions of the protein. Identical peptide maps were generated from striatum- and nucleus accumbens-derived transporters, indicating that these polypeptides are highly similar in primary sequence. The proteolytic fragments generated by V8 protease were localized to specific domains of the protein using antipeptide antibodies corresponding to five different regions of the transporter. Fragments of 10 and 7 kDa from [125I]DEEP-labeled transporters were specifically immunoprecipitated with an antibody generated against amino acids 42-59 (near the first putative trans-membrane domain), whereas a 34-kDa fragment from [125I]RTI 82-labeled transporters was precipitated with three different sera corresponding to regions in the carboxyl-terminal two thirds of the protein. None of the V8 fragments smaller than 45 kDa, containing either photolabel, was altered in molecular mass by N-deglycosylation. The results indicate that photoincorporation of [125I]DEEP occurs in the amino half of the dopamine transporter, near the first two transmembrane helices, whereas [125I]RTI 82 labels the carboxyl-terminal region of the protein, between transmembrane domains 4 and 12.
In vivo electrochemistry was used to determine the effects of locally applied raclopride (a D2 receptor antagonist) and SCH-23390 (a D1 receptor antagonist) on the clearance of locally applied dopamine in the striatum, nucleus accumbens, and medial prefrontal cortex of rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated, single carbon fiber electrodes. When a calibrated amount of dopamine was pressure ejected at 5-min intervals from a micropipette adjacent (280-310 microns) to the electrode, transient and reproducible dopamine signals were detected in all three regions. Local application of raclopride from a second micropipette, prior to pressure ejection of dopamine, increased the amplitude and time course of the dopamine signals, indicating significant inhibition of the dopamine transporter. In contrast, local application of SCH-23390 or saline had no effect on the dopamine signals. These data indicate that D2, but not D1, dopamine receptors can modulate the activity of the dopamine transporter.
Dysfunction of dopamine neural systems is hypothesized to underlie neuropsychiatric disorders and psychostimulant drug abuse. At least three dopamine systems have been characterized in the brain-nigrostriatal, mesolimbic, and mesocortical. Abnormalities of nigrostriatal dopamine neurons cause motor impairment leading to Parkinson's disease, whereas dysfunction of mesolimbic and mesocortical dopamine neurons are most implicated in psychotic disorders such as schizophrenia and in drug addition. One of the primary neural sites of action of potent antipsychotic agents and psychostimulant drugs of abuse are dopamine receptors and dopamine transporters which, respectively, mediate the induction and termination of dopamine's actions. Very limited information is, however, available about which particular set of dopaminergic cells in the human brain actually express the genes for these dopamine-specific proteins. In this study, we observed that the dopamine transporter and D2 receptor messenger RNAs are differentially expressed within the human mesencephalon: highest expression in ventral subpopulations of the substantia nigra pars compacta neurons with lowest expression in the mesolimbic/mesocortical ventral tegmental area and retrorubral cell groups. These findings suggest that motor- and limbic-related mesencephalic neurons in the human brain differ in the degree of dopamine transporter and D2 receptor gene expression.
Chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to baboons was shown previously to result in a motor syndrome and a pattern of striatal dopaminergic fibre loss similar to those observed in idiopathic Parkinson's disease. In the present study, tyrosine hydroxylase-immunoreactive neurons were quantified in the mesencephalon of control (n = 4) and chronically MPTP-treated (n = 3) baboons. MPTP induced a significant reduction in neuronal cell density in the substantia nigra (63.8% reduction) and the ventral tegmental area (53.1%). Within the substantia nigra, obvious mediolateral and dorsoventral gradients of neuronal cell loss were observed. First, the pars lateralis was more affected than the lateral divisions of the pars compacta (89.6% vs 73.8% cell loss), which in turn were more depleted than the medial divisions (60.1% reduction). Second, the ventral regions of the pars compacta were more degenerated than the dorsal parts (82.4 vs 51.5% decrease). This regional pattern is strikingly similar to that observed in Parkinson's disease and indicates that two subpopulations of dopaminergic neurons are distinguishable on the basis of their differential vulnerability to MPTP. Finally, the present study confirms that chronic mitochondrial complex I inhibition using MPTP in primates is sufficient to reproduce the typical dopaminergic cell loss and striatal fibre depletion observed in Parkinson's disease.
Dopamine transporter messenger RNA (mRNA) expression was assessed by in situ hybridization over individual pigmented neurons from the substantia nigra pars compacta in midbrain sections from 7 parkinsonian and 7 age-matched, neurologically normal patients. In the normal control brains, high levels of expression of dopamine transporter mRNA were noted over pigmented neurons in the substantia nigra pars compacta; neurons in the adjacent nucleus paranigralis of the ventral tegmental area displayed less hybridization. Nigra compacta neurons surviving in brains of patients with Parkinson's disease displayed only 57% of the dopamine transporter mRNA hybridization intensity displayed by nigral neurons in normal control brains. The disease-related decrease in the apparent level of dopamine transporter mRNA expression in remaining neurons could reflect neuronal dysfunction. Conceivably, it might also reflect differential vulnerability of those neurons that initially expressed higher levels of this transporter to the insult of parkinsonism.
During the last decade, there have been enormous advances in our knowledge of glycoproteins and the stage has been set for the biotechnological production of many of them for therapeutic use. These advances are reviewed, with special emphasis on the structure and function of the glycoproteins (excluding the proteoglycans). Current methods for structural analysis of glycoproteins are surveyed, as are novel carbohydrate-peptide linking groups, and mono- and oligo-saccharide constituents found in these macromolecules. The possible roles of the carbohydrate units in modulating the physicochemical and biological properties of the parent proteins are discussed, and evidence is presented on their roles as recognition determinants between molecules and cells, or cell and cells. Finally, examples are given of changes that occur in the carbohydrates of soluble and cell-surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.
The apparent molecular masses of photoaffinity-labeled dopamine transporters (DATs) from rat, human, dog, and primate kidney COS cells expressing the rat DAT1 cDNA differ. Sequences predicted from cDNA cloning reveal only one amino acid difference between the length of the rat and human DAT but one less site for potential N-linked glycosylation in the human DAT. Possible posttranslational and postmortem bases for species differences in DAT molecular mass were explored. Rat DAT proteins from striata subjected to approximately 5 h of postmortem delay modeled after the human postmortem delay process revealed small but consistent losses in apparent molecular mass and in cocaine analogue binding; the DAT molecular mass displayed no further losses for up to 30 h of model postmortem treatment. Degradative postmortem changes could thus contribute to molecular mass differences between rat and human DATs. Neuraminidase treatment reduced the apparent molecular mass of native rat DAT but not that of the rat DAT expressed in COS cells, suggesting that the sugars added to the DAT expressed in COS cells were different than those added to the rat brain striatal transporter. These differences could account for the somewhat higher Km values for expressed DAT cDNA in COS cells when compared with the wild-type striatal transporter. These results are in accord with the differences in number of predicted N-linked glycosylation sites between rat and human DATs and with cell-type specificity in transporter posttranslational processing.
Dopamine transporter mRNA expression in individual neurons from the substantia nigra pars compacta. 'All' area, arcuate nucleus of the hypothalamus, retina, and olfactory bulb was assessed by in situ hybridization. High levels of expression were noted over individual neurons in midbrain nuclei; much lower expression was found in cells of the inner nuclear layer of the retina, glomerular cell layer of the olfactory bulb, and medial aspect of the arcuate nucleus of the hypothalamus. The low levels of expression in the latter nuclei are consistent with the paucity of effects of cocaine in visual and olfactory systems, failure to detect photoaffinity-labelled transporter protein in hypothalamus or olfactory bulb, and observations that little or no damage is found in dopaminergic neurons outside the basal midbrain in idiopathic Parkinsonism.
Neurotransmission, which represents chemical signalling between neurons, usually takes place at highly differentiated anatomical structures called synapses. To fulfill both the time and space confinements required for optimal neurotransmission, highly specialized proteins, known as transporters or uptake sites, occur and operate at the presynaptic plasma membrane. Using the energy provided by the Na+ gradient generated by the Na+/K(+)-transporting ATPase, these transporters reuptake the neurotransmitters soon after their release, thereby regulating their effective concentrations at the synaptic cleft and the availability of neurotransmitters for a time-dependent activation of both pre- and postsynaptic receptors. The key role these proteins play in normal neurotransmission is further emphasized when the physiological and social consequences of drugs that interfere with the function of these transporters, such as the psychostimulants (e.g. amphetamine and cocaine) or the widely prescribed antidepressant drugs, are considered. In this review, Bruno Giros and Marc Caron elaborate on the potential consequences of the recent molecular cloning of the dopamine and related transporters and summarize some of the interesting properties that are emerging from this growing family of Na(+)- and Cl(-)-dependent transporters.