Effects of VMAT2 inhibitors lobeline and GZ‐793A on methamphetamine‐induced changes in dopamine release,metabolism and synthesis in vivo

Vesicular monoamine transporter‐2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N‐(1,2R‐dihydroxylpropyl)‐2,6‐cis‐di(4‐methoxyphenethyl)piperidine hydrochloride (GZ‐793A; 15 or 30 mg/kg) on METH‐induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward‐relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ‐793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time‐dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ‐793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ‐793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH‐induced increase in extracellular DA. Both LOB and GZ‐793A enhanced the duration of the METH‐induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ‐793A decreased synthesis; no effect of METH or GZ‐793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time‐dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ‐793A to decrease METH reward.

     

Iron Deficiency Alters the Day‐Night Variation in Monoamine Levels in Mice

Monoamine metabolism in the central nervous system is altered by dietary iron deficiency, with a stronger effect seen during the active than rest span of the circadian cycle. In this report, we examined changes in intracellular and extracellular monoamine levels, synthetic enzymes, transporter and receptor densities, and responses to amphetamine‐induced dopamine (DA) efflux in iron‐deficient and iron‐sufficient mice. Extracellular striatal DA levels were 15–20% higher in all groups during the active dark phase compared to the inactive light phase, with correspondingly lower dopamine transporter (DAT) and higher tyrosine hydroxylase levels. Iron deficiency decreased DAT density by 20% and 28% in the light and dark phases, respectively, and elevated the DOPAC/DA ratio only in the dark, indicating that iron deficiency does interact with the normal diurnal cues for cyclicity. Enhanced DA efflux after amphetamine stimulation indicates no limitation on monoamine synthesis and release and is consistent with altered synaptic efficacy and perhaps recycling of DA in iron deficiency. These experimental findings provide new evidence that brain iron insufficiency does have a differential effect on the DA system at different biological times of the day and night and may be causally related to the phasic motor symptoms observed in Restless Legs Syndrome.     

Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5–10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.     

The Selective Inhibition of the D1 Dopamine Receptor Results in an Increase of Metabolized Dopamine in the Rat Striatum

Our aim was to study the specific role of the postsynaptic D(1) receptors on dopaminergic response and analyze the metabolized dopamine (DA) in the rat striatum. We used male Wistar rats to evaluate the effects of different doses of a D(1) agonist (SKF-38393) and a D(1) antagonist (SCH-23390), and their co-administration. The levels of DA and L-3, 4-dihydroxyphenylacetic acid (DOPAC) were measured using high performance liquid chromatography. The systemic injection of SKF-38393 alone at 1, 5 and 10 mg/kg did not alter the DA and DOPAC levels or the DOPAC/DA ratio. In contrast, injection of SCH-23390 alone at 0.25, 0.5 and 1 mg/kg significantly increased the DA and DOPAC levels, as well as the DOPAC/DA ratio, compared with the respective control groups. The co-administration of SCH-23390+SKF-38393 did not alter the DA or DOPAC levels, but it did significantly inhibit the SCH-23390-induced increase of the DA and DOPAC levels. The SCH-23390+SKF-38393 and the SCH-23390-only groups showed an increase in the DOPAC/DA ratio. The co-administration of SCH-23390+PARGYLINE significantly decreased the DOPAC levels and the DOPAC/DA ratio compared with the control and SCH-23390 groups. Taken together, our results showed that selective inhibition with SCH-23390 produced an increase in metabolized DA via striatal monoamine oxidase. These findings also contribute to the understanding of the role of postsynaptic D(1) receptors in the long-loop negative feedback system in the rat striatum.     

An In Vivo Study of Dopamine Release and Metabolism in Rat Brain Regions Using Intracerebral Dialysis

Intracerebral dialysis was used with a specifically designed HPLC with electrochemical detection assay to monitor extracellular levels of endogenous 3,4-dihydroxyphenylethylamine (dopamine, DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in brain regions of the halothane-anesthetized rat. Significant amounts of DA, DOPAC, and HVA were detected in control perfusates collected from striatum and n. accumbens whereas the medial prefrontal cortex showed lower monoamine levels. The ratio of DA in perfusate to DA in whole tissue suggests that in f. cortex, compared to n. accumbens and striatum, there is a greater amount of DA in the extracellular space relative to the intraneuronal DA content. The DOPAC/HVA ratio in control perfusates varied between regions in accordance with whole tissue measurements. This ratio was highest in n. accumbens and lowest in f. cortex. The monoamine oxidase inhibitor pargyline (100 mg/kg i.p.) caused an exponential decline in DOPAC, but not of HVA, in regional perfusates, an effect that was associated with an increase in DA. The data indicated a higher turnover of extracellular DOPAC in n. accumbens than in striatum and the lowest DOPAC turnover in f. cortex. The rate of decline in extracellular DA metabolite levels was slow compared to whole tissue measurements. In the perfusates there was no statistical correlation between basal amounts of DA in the perfusates and DOPAC and HVA levels or DOPAC turnover for any of the areas, indicating that measurement of DA metabolism in the brain under basal conditions does not provide a good index of DA release. In summary, this study shows clear regional differences in basal DA release and metabolite levels, metabolite patterns, and DOPAC turnover rates in rat brain in vivo.     

The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells

The NADH shuttle system, which transports reducing equivalents from the cytosol to the mitochondria, is essential for the coupling of glucose metabolism to insulin secretion in pancreatic beta cells. Aralar1 and citrin are two isoforms of the mitochondrial aspartate/glutamate carrier, one key constituent of the malate-aspartate NADH shuttle. Here, the effects of Aralar1 overexpression in INS-1E beta cells and isolated rat islets were investigated for the first time. We prepared a recombinant adenovirus encoding for human Aralar1 (AdCA-Aralar1), tagged with the small FLAG epitope. Transduction of INS-1E cells and isolated rat islets with AdCA-Aralar1 increased aralar1 protein levels and immunostaining revealed mitochondrial localization. Compared with control INS-1E cells, overexpression of Aralar1 potentiated metabolism secretion coupling stimulated by 15 mm glucose. In particular, there was an increase of NAD(P)H generation, of mitochondrial membrane hyperpolarization, ATP levels, glucose oxidation, and insulin secretion (+45%, p < 0.01). Remarkably, this was accompanied by reduced lactate production. Rat islets overexpressing Aralar1 secreted more insulin at 16.7 mm glucose (+65%, p < 0.05) compared with controls. These results show that aspartate-glutamate carrier capacity limits glucose-stimulated insulin secretion and that Aralar1 overexpression enhances mitochondrial metabolism.     

Determinants of buildup of the toxic dopamine metabolite DOPAL in Parkinson's disease

Intra‐neuronal metabolism of dopamine (DA) begins with production of 3,4‐dihydroxyphenylacetaldehyde (DOPAL), which is toxic. According to the ‘catecholaldehyde hypothesis,’ DOPAL destroys nigrostriatal DA terminals and contributes to the profound putamen DA deficiency that characterizes Parkinson's disease (PD). We tested the feasibility of using post‐mortem patterns of putamen tissue catechols to examine contributions of altered activities of the type 2 vesicular monoamine transporter (VMAT2) and aldehyde dehydrogenase (ALDH) to the increased DOPAL levels found in PD. Theoretically, the DA : DOPA concentration ratio indicates vesicular uptake, and the 3,4‐dihydroxyphenylacetic acid : DOPAL ratio indicates ALDH activity. We validated these indices in transgenic mice with very low vesicular uptake (VMAT2‐Lo) or with knockouts of the genes encoding ALDH1A1 and ALDH2 (ALDH1A1,2 KO), applied these indices in PD putamen, and estimated the percent decreases in vesicular uptake and ALDH activity in PD. VMAT2‐Lo mice had markedly decreased DA:DOPA (50 vs. 1377, p < 0.0001), and ALDH1A1,2 KO mice had decreased 3,4‐dihydroxyphenylacetic acid:DOPAL (1.0 vs. 11.2, p < 0.0001). In PD putamen, vesicular uptake was estimated to be decreased by 89% and ALDH activity by 70%. Elevated DOPAL levels in PD putamen reflect a combination of decreased vesicular uptake of cytosolic DA and decreased DOPAL detoxification by ALDH.

     

Vesicular monoamine transporter 2 mediates fear behavior in mice

A subset of people exposed to a traumatic event develops post‐traumatic stress disorder (PTSD), which is associated with dysregulated fear behavior. Genetic variation in SLC18A2, the gene that encodes vesicular monoamine transporter 2 (VMAT2), has been reported to affect risk for the development of PTSD in humans. Here, we use transgenic mice that express either 5% (VMAT2‐LO mice) or 200% (VMAT2‐HI mice) of wild‐type levels of VMAT2 protein. We report that VMAT2‐LO mice have reduced VMAT2 protein in the hippocampus and amygdala, impaired monoaminergic vesicular storage capacity in both the striatum and frontal cortex, decreased monoamine metabolite abundance and a greatly reduced capacity to release dopamine upon stimulation. Furthermore, VMAT2‐LO mice showed exaggerated cued and contextual fear expression, altered fear habituation, inability to discriminate threat from safety cues, altered startle response compared with wild‐type mice and an anxiogenic‐like phenotype, but displayed no deficits in social function. By contrast, VMAT2‐HI mice exhibited increased VMAT2 protein throughout the brain, higher vesicular storage capacity and greater dopamine release upon stimulation compared with wild‐type controls. Behaviorally, VMAT2‐HI mice were similar to wild‐type mice in most assays, with some evidence of a reduced anxiety‐like responses. Together, these data show that presynaptic monoamine function mediates PTSD‐like outcomes in our mouse model, and suggest a causal link between reduced VMAT2 expression and fear behavior, consistent with the correlational relationship between VMAT2 genotype and PTSD risk in humans. Targeting this system is a potential strategy for the development of pharmacotherapies for disorders like PTSD.     

Regional brain monoamine levels and utilization in middle-aged rats.

Significant changes in monoamine levels and utilization were noted in certain brain regions of middle-aged Fisher 344 rats when compared with young adult controls. In the prefrontal cortex and septum, 3,4 dihydroxyphenylglycol (MHPG) and the MHPG/norepinephrine (NE) ratio were decreased. The septum also showed increases in dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) but there was a decrease in the DOPAC/DA ratio. The striatum showed an increase in the MPHG/NE ratio and an increase in DOPAC. The hippocampus and thalamus showed an increase in 5-hydroxyindoleacetic acid (5HIAA). This demonstrates that selected neurotransmitter systems in the brain are altered at an early stage of senescence. This could lead to ensuing neurological deficits.     

Attenuation of estradiol on the reduction of striatal dopamine by amphetamine in ovariectomized rats

Amphetamine (AMPH) is a highly addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long‐term abusers. Estrogen seems to show neuroprotection in dopamine (DA) deficit caused by AMPH. The present study was to investigate the effects of estradiol on the levels of striatal DA in ovariectomized (Ovx) rats treated with or without AMPH. Female rats were Ovx for 2 weeks before administration of AMPH (5 mg/kg/day, i.p.) with or without 17β‐estradiol benzoate (EB) (25 µg/kg/day, s.c.) for 7 days. The striatal tissues were collected, homogenized with DA mobile phase, and centrifuged. The concentrations of DA in the supernatants were detected by HPLC. The protein expressions of dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT‐2), and tyrosine hydroxylase (TH) were analyzed by Western blotting. The results indicated that AMPH could attenuate DA level significantly in striatum (P < 0.01). Comparing to control groups, administration of either EB or EB plus AMPH increased DA level (P < 0.01). The protein expression of striatal DAT was significant greater (P < 0.01) in rats treated with AMPH plus EB than AMPH treated animals. These results suggest that the DA levels in striatum can be enhanced by EB via an increase of DAT expression following administration of AMPH. J. Cell. Biochem. 108: 1318–1324, 2009. © 2009 Wiley‐Liss, Inc.     

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1–<Superscript>13</Superscript>C)-Glucose

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1–¹³C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30–35?%) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.     

A Metabolomic Study of Brain Tissues from Aged Mice with Low Expression of the Vesicular Monoamine Transporter 2 (VMAT2) Gene

The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles in preparation for neurotransmission. Samples of cerebellum, cortex, hippocampus, substantia nigra and striatum from VMAT2-deficient mice were compared to age-matched control mice. Multivariate statistical analyses of ¹H NMR spectral profiles separated VMAT2-deficient mice from controls for all five brain regions. Although the data show that metabolic alterations are region- and age-specific, in general, analyses indicated decreases in the concentrations of taurine and creatine/phosphocreatine and increases in glutamate and N-acetyl aspartate in VMAT2-deficient mouse brain tissues. This study demonstrates the efficacy of metabolomics as a functional genomics phenotyping tool for mouse models of neurological disorders, and indicates that mild reductions in the expression of VMAT2 affect normal brain metabolism. Special issue article in honor of Dr. Frode Fonnum.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.     

Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum

Because age-related changes in brain dopaminergic innervation are assumed to influence human disorders involving dopamine (DA), we measured the levels of several presynpatic DAergic markers [DA, homovanillic acid, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT)] in post-mortem human striatum (caudate and putamen) from 56 neurologically normal subjects aged 1 day to 103 years. Striatal DA levels exhibited pronounced (2- to 3-fold) post-natal increases through adolescence and then decreases during aging. Similarly, TH and AADC increased almost 100% during the first 2 post-natal years; however, the levels of TH and, to a lesser extent, AADC then declined to adult levels by approximately 30 years of age. Although VMAT2 and DAT levels closely paralleled those of TH, resulting in relatively constant TH to transporter ratios during development and aging, a modest but significant decline (13%) in DAT levels was observed in only caudate during aging. This biphasic post-natal pattern of the presynaptic markers suggests that striatal DAergic innervation/neuropil appears to continue to develop well past birth but appears to become overelaborated and undergo regressive remodeling during adolescence. However, during adulthood, a striking discrepancy was observed between the loss of DA and the relative preservation of proteins involved in its biosynthesis and compartmentation. This suggests that declines in DA-related function during adulthood and senescence may be explained by losses in DA per se as opposed to DAergic neuropil.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Turnover of Dopamine and Dopamine Metabolites in Rat Brain: Comparison Between Striatum and Substantia Nigra

Measurements of the turnover of dopamine (DA) and DA metabolites have been performed in the striatum and substantia nigra (SN) of the rat. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid have been assessed from the disappearance rates after blocking their formation by inhibition of monoamine oxidase by pargyline and of catechol-O-methyltransferase by tropolone. DA turnover has been measured as 3-methoxytyramine (3-MT) plus DA accumulation rate after MAO inhibition by pargyline and as accumulation rate of 3,4-dihydroxyphenylalanine (DOPA) after inhibition of aromatic amino acid decarboxylase by NSD 1015 or NSD 1034. These measures of DA turnover have been compared with alpha-methyl-p-tyrosine (alpha-MT)-induced DA disappearance rate. In SN all the different measures of DA turnover are in the same range (55-62 nmol/g protein/h) whereas in striatum DOPA accumulation rate after NSD 1015 and alpha-MT-induced DA disappearance rate (16-23 nmol/g/h) are much lower than DOPAC disappearance rate after pargyline, 3-MT plus DA accumulation rate after pargyline, and DOPA accumulation rate after NSD 1034 (39-46 nmol/g/h). The data confirm our previous findings indicating that the fractional turnover rate of DA is more rapid in SN than in striatum and that O-methylation of DA is relatively more important in SN. In striatum at least two pools of DA with different turnover rates appear to exist, whereas in SN, DA behaves as if located in a single compartment.     

Altered expression of key dopaminergic regulatory proteins in the postnatal brain following perinatal n-3 fatty acid dietary deficiency

The consequences of maternal linolenic acid (LNA, 18:3n-3) dietary deficiency on key dopamine (DA)-associated regulatory proteins in mesolimbic and mesocortical structures of the postnatal rat brain have been investigated. A marked (4.5-fold) decrease of the DA-synthesizing enzyme tyrosine hydroxylase accompanied by a down-regulation (approx 7.5-fold) of the vesicular monoamine transporter (VMAT-2) and a depletion of VMAT-associated vesicles in the hippocampus were observed in deficient offspring compared with adequately fed controls. The DA transporter (DAT) was not affected by the LNA deficiency indicative of a DAT/VMAT-2 ratio increase that may enhance the risk of damage of the dopaminergic (DAergic) terminal. A robust increase in DA receptor (DAR1 and DAR2) levels was noticed in the cortex and striatum structures possibly to compensate for the low levels of DA in synaptic clefts. Microglia activation characterized by enhanced levels of ED1 antibody and nuclear internalization of p65 NFκB was noticed following LNA deficiency. Diminished levels of 22:6n-3 docosahexaenoic acid ( Schiefermeier and Yavin 2002 ), the most ubiquitous metabolite generated by LNA is proposed to reduce the anti-oxidant arsenal in the developing brain and cause microglia activation and enhanced oxidative stress to increase the risk of certain DA-associated neurological disorders.     

β‐Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion

We evaluated the neuroprotective effects of β‐methylphenylalanine in an experimental model of rotenone‐induced Parkinson's disease (PD) in SH‐SY5Y cells and rats. Cells were pre‐treated with rotenone (2.5 µg/mL) for 24 hours followed by β‐methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone–pre‐treated cells, β‐methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. β‐Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH‐SY5Y cells. In the rotenone‐induced rat model of PD, oral administration of β‐methylphenylalanine recovered DA and DOPAC levels and bradykinesia. β‐Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and β‐methylphenylalanine. Our experimental results show neuroprotective effects of β‐methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that β‐methylphenylalanine may be useful in the treatment of PD.