Toxic effects of human and rodent variants of alpha-synuclein in vivo.

In Parkinson's disease, abnormal alpha-synuclein (asyn) accumulation leads to the formation of soluble oligomeric species thought to be toxic to cells as well as intraneuronal inclusions. To date, the precise mechanisms leading to aggregation of asyn in the brain is not well-understood. Previous studies in yeast, drosophila, and transgenic mice suggested that a non-A beta component depleted version of human asyn [h-asyn(D70-83)] or human beta-synuclein (h-bsyn), naturally lacking this centrally located hydrophobic region, are less prone to form aggregates in vitro and are expected to be less toxic compared to h-asyn in vivo, although not all experimental studies unequivocally support the latter view. To address this outstanding issue, we directly compared the neurotoxicity of human asyn against that of h-asyn(D70-83), h-bsyn as well as rat asyn using an adeno-associated viral vector to express these proteins in a dose-response study where the vector load was varied over two orders of magnitude. By quantifying the neurodegeneration of rat substantia nigra dopamine neurons here we show that h-asyn, h-bsyn, and h-asyn(D70-83) display comparable neurotoxicity across the vector doses tested. On the other hand, rat asyn and GFP control vectors displayed a different profile, where no detectable neurodegeneration was seen except at the highest vector titer. Thus, the two main conclusions of our study are that (i) deletion of the central hydrophobic region in h-asyn is not sufficient to alter its neurotoxic properties and (ii) expression of the widely used GFP control protein can cause measurable neurodegeneration at high titers.

Assessment of brain metabolite correlates of adeno-associated virus-mediated over-expression of human alpha-synuclein in cortical neurons by in vivo (1) H-MR spectroscopy at 9.4 T.

In this study, we used proton-localized spectroscopy ((1) H-MRS) for the acquisition of the neurochemical profile longitudinally in a novel rat model of human wild-type alpha-synuclein (α-syn) over-expression. Our goal was to find out if the increased α-syn load in this model could be linked to changes in metabolites in the frontal cortex. Animals injected with AAV vectors encoding for human α-syn formed the experimental group, whereas green fluorescent protein expressing animals were used as the vector-treated control group and a third group of uninjected animals were used as naïve controls. Data were acquired at 2, 4, and 8 month time points. Nineteen metabolites were quantified in the MR spectra using LCModel software. On the basis of 92 spectra, we evaluated any potential gender effect and found that lactate (Lac) levels were lower in males compared to females, while the opposite was observed for ascorbate (Asc). Next, we assessed the effect of age and found increased levels of GABA, Tau, and GPC+PCho. Finally, we analyzed the effect of treatment and found that Lac levels (p = 0.005) were specifically lower in the α-syn group compared to the green fluorescent protein and control groups. In addition, Asc levels (p = 0.05) were increased in the vector-injected groups, whereas glucose levels remained unchanged. This study indicates that the metabolic switch between glucose-lactate could be detectable in vivo and might be modulated by Asc. No concomitant changes were found in markers of neuronal integrity (e.g., N-acetylaspartate) consistent with the fact that α-syn over-expression in cortical neurons did not result in neurodegeneration in this model. We acquired the neurochemical profile longitudinally in a rat model of human wild-type alpha-synuclein (α-syn) over-expression in cortical neurons. We found that Lactate levels were reduced in the α-syn group compared to the control groups and Ascorbate levels were increased in the vector-injected groups. No changes were found in markers of neuronal integrity consistent with the fact that α-syn over-expression did not result in frank neurodegeneration.

Ser129 phosphorylation of endogenous α-synuclein induced by overexpression of polo-like kinases 2 and 3 in nigral dopamine neurons is not detrimental to their survival and function.

Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-syn] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase (PLK) 2 or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.

Dysregulated dopamine storage increases the vulnerability to α-synuclein in nigral neurons.

Impairments in the capacity of dopaminergic neurons to handle cytoplasmic dopamine may be a critical factor underlying the selective vulnerability of midbrain dopamine neurons in Parkinson's disease. Furthermore, toxicity of α-synuclein in dopaminergic neurons has been suggested to be mediated by direct interaction between dopamine and α-synuclein through formation of abnormal α-synuclein species, although direct in vivo evidence to support this hypothesis is lacking. Here, we investigated the role of dopamine availability on α-synuclein mediated neurodegeneration in vivo. We found that overexpression of α-synuclein in nigral dopamine neurons in mice with deficient vesicular storage of dopamine led to a significant increase in dopaminergic neurodegeneration. Importantly, silencing the tyrosine hydroxylase enzyme - thereby reducing dopamine content in the nigral neurons - reversed the increased vulnerability back to the baseline level observed in wild-type littermates, but failed to eliminate it completely. Importantly, TH knockdown was not effective in altering the toxicity in the wild-type animals. Taken together, our data suggest that under normal circumstances, in healthy dopamine neurons, cytoplasmic dopamine is tightly controlled such that it does not contribute significantly to α-synuclein mediated toxicity. Dysregulation of the dopamine machinery in the substantia nigra, on the other hand, could act as a trigger for induction of increased toxicity in these neurons and could explain how these neurons become more vulnerable and die in the disease process.

The alpha(2) adrenoceptor antagonist idazoxan alleviates L-DOPA-induced dyskinesia by reduction of striatal dopamine levels: an in vivo microdialysis study in 6-hydroxydopamine-lesioned rats.

L-DOPA-induced dyskinesia is characterised by debilitating involuntary movement, which limits quality of life in patients suffering from Parkinson's disease. Here, we investigate effects of the a2 adrenoceptor antagonist idazoxan on L-DOPA-induced dyskinesia as well as on alterations of extracellular L-DOPA and dopamine (DA) levels in the striatum in dyskinetic rats. Male Wistar rats were unilaterally lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA/benserazide to induce stable dyskinetic movements.Administration of idazoxan [(9 mg/kg, intraperitoneal (i.p.)]significantly alleviated L-DOPA-induced dyskinesia, whereas idazoxan (3 mg/kg, i.p.) did not affect dyskinetic behaviour.Bilateral in vivo microdialysis revealed that idazoxan 9 mg/kg reduces extracellular peak L-DOPA levels in the lesioned and intact striatum as well as DA levels in the lesioned striatum. In parallel, the exposure to idazoxan in the striatum was monitored.Furthermore, no idazoxan and L-DOPA drug-drug interaction was found in plasma, brain tissue and CSF. In conclusion, the decrease of L-DOPA-derived extracellular DA levels in the lesioned striatum significantly contributes to the anti-dyskinetic effect of idazoxan.

The selective alpha1 adrenoceptor antagonist HEAT reduces L-DOPA-induced dyskinesia in a rat model of Parkinson's disease.

In Parkinson's disease (PD), the long term use of L-DOPA results in major adverse effects including dyskinesia or abnormal involuntary movements. The present study focuses on the effect of the selective alpha(1) adrenoceptor antagonist HEAT (2-[[beta-(-4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) in the 6-hydroxydopamine rat model of L-DOPA-induced dyskinesia. We demonstrate that the selective alpha(1) adrenoceptor antagonist HEAT (1 and 2 mg kg(-1)), the alpha(2) adrenoceptor antagonist idazoxan (9 mg kg(-1)), and the nonselective beta(1)/beta(2) adrenoceptor antagonist propranolol (20 mg kg(-1)) alleviate dyskinetic movements induced by L-DOPA. Furthermore, the adrenoceptor antagonists at the doses used did not influence exploratory behavior in the open field system indicating that the antidyskinetic effect is not due to a reduction in general motor activity. Intrastriatal administration of the selective alpha(1) adrenoceptor agonist cirazoline via reverse in vivo microdialysis did not induce dyskinesia. Additionally, we measured plasma, brain, and CSF levels of HEAT. HEAT is a CNS active compound with a brain/plasma and CSF/plasma ratio of 4.29 and 0.15, respectively, which is appropriate for the investigation of alpha(1)-mediated mechanisms in CNS disorders. In conclusion, these results demonstrated for the first time that a alpha(1) adrenoceptor antagonist reduced L-DOPA-induced dyskinesia in a rat model. Further studies assessing the risk benefit in comparison to existing therapies are needed before considering alpha(1) adrenoceptor antagonists as a target for the development of new antidyskinetic compounds.

Continuous dopaminergic stimulation by pramipexole is effective to treat early morning akinesia in animal models of Parkinson's disease: A pharmacokinetic-pharmacodynamic study using in vivo microdialysis in rats.

Short-acting dopamine (DA) agonists are usually administered several times a day resulting in fluctuating plasma and brain levels. DA agonists providing continuous dopaminergic stimulation may achieve higher therapeutic benefit for example by alleviating nocturnal disturbances as well as early morning akinesia. In the present study continuous release (CR) of pramipexole (PPX) was maintained by subcutaneous implantation of Alzet minipumps, whereas subcutaneous PPX injections were used to mimic PPX immediate release (IR) in male Wistar rats. In the catalepsy bar test, PPX-CR (1 mg/kg/day) reversed the haloperidol-induced motor impairment in the morning and over the whole observation period of 12h. In contrast, PPX-IR (tid 1 mg/kg, pre-treatment the day before) was not effective in the morning but catalepsy was reduced for 6 h after PPX-IR (1 mg/kg) injection. In the reserpine model, early morning akinesia indicated by the first motor activity measurement in the morning was significantly reversed by PPX-CR (2 mg/kg/day). Again, PPX-IR (tid 0.3 mg/kg, pre-treatment the day before) was not able to antagonise early morning akinesia. These results are in agreement with in vivo microdialysis measurements showing a continuous decrease of extracellular DA levels and a continuous PPX exposure in the PPX-CR (1 mg/kg/day) group. In contrast, PPX-IR (0.3 mg/kg) produced a transient decrease of extracellular DA levels over 6 h and showed maximum PPX levels 2 h after dosing which decreased over the following 6-8 h. The present study demonstrates that PPX-CR may offer a higher therapeutic benefit than PPX-IR on early morning akinesia and confirms earlier reports that PPX-IR reverses motor impairment for several hours.

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein.

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

Effects of levodopa on striatal monoamines in mice with levodopa-induced hyperactivity.

The present study examines striatal monoamine changes in a murine model of levodopa-induced dyskinesia (LID), a common side effect of Parkinson's disease (PD) therapy. Mice previously exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and later made hyperactive with high-dose (200mg/kg, i.p.) exogenous levodopa were compared to mice with normal motor behavior who received either levodopa without previous MPTP or no treatment at all. Using high-performance liquid chromatography, dopamine (DA), serotonin (5HT), noradrenaline (NA) and their metabolites were then measured in samples of striatum versus olfactory bulbs as controls. In the olfactory bulb, exogenous levodopa caused increased DA levels and increased DA-, 5HT- and NA-turnover rates, but decreased 5HT and NA levels, regardless of animal activity. These trends were also seen in the striatum, but animals with LID seemed to have unique differences. Thus, in mice sacrificed at the height of their hyperactive LID behavior, striatal DA and 5HT were significantly lower and DA- and 5HT-turnover rates were significantly higher than control animals with normal motor behavior, regardless of levodopa exposure. In addition, the expected increased NA-turnover rate seen in other specimens from animals exposed to levodopa was not seen in the striatum of LID mice. The results of the present study demonstrate that there is a distinct profile of striatal monoamines conducive to LID that must be considered when trying to explain the effects of anti-LID drugs utilizing monoamine receptors.

L-DOPA-induced dyskinesia in Parkinson's disease: a drug discovery perspective.

L-DOPA-induced dyskinesia is a debilitating, treatment-limiting adverse effect that eventually occurs in the majority of Parkinson's disease patients. This review describes the strategy of biological testing, focusing on in vivo proof-of-concept animal models. We distinguish between symptomatic efficacy markers in predictive preclinical in vivo models and confounding factors that eventually produce false positive results. Clinical studies and predicted efficacy from preclinical studies are recapitulated and insights into the attempt to bridge preclinical and clinical studies are given. Furthermore, the mode of action that is to be involved in emerging therapy against L-DOPA-induced dyskinesia is reviewed.

Rapid analysis of GABA and glutamate in microdialysis samples using high performance liquid chromatography and tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been established for the rapid and reliable determination of gamma-aminobutyric acid (GABA) and glutamate in brain microdialysates. The microdialysis samples were analysed using a HILIC (hydrophilic interaction liquid chromatography) column, which is able to retain the polar amino acid neurotransmitters. The mobile phase consisted of a binary gradient elution profile comprising 0.1% formic acid in water and acetonitrile. GABA, glutamate as well as the respective internal standards [D(6)]-GABA and [D(5)]-glutamate were detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation mode within a running time of 3 min. The linearity ranged from 1 nM to 10 microM for GABA and 10 nM to 10 microM for glutamate. The limit of quantitation was found to be 1 nM for GABA and 10nM for glutamate (injection volume 10 microl). The present LC-MS/MS method was compared to the classical method for analysis of GABA and glutamate using high performance liquid chromatography (HPLC) and fluorescence detection (FD). Eventually, the feasibility of the LC-MS/MS method was demonstrated using in vivo microdialysis in rats by monitoring changes of the extracellular concentrations of GABA and glutamate in the globus pallidus following stimulation with potassium.

Intrastriatal inhibition of aromatic amino acid decarboxylase prevents l-DOPA-induced dyskinesia: a bilateral reverse in vivo microdialysis study in 6-hydroxydopamine lesioned rats.

l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia consists of involuntary choreiform and dystonic movements. Here we report whether intrastriatal l-DOPA itself is able to trigger dyskinetic behavior and which role the neurotransmitter dopamine (DA) and its metabolites play. Intrastriatal l-DOPA as well as DA administration at the 6-hydroxydopamine (6-OHDA) lesioned side led to a significant appearance of dyskinetic behavior, whereas DA metabolites were ineffective. Intrastriatal inhibition of the enzyme aromatic amino acid decarboxylase (AADC) by benserazide prevented the appearance of l-DOPA-induced dyskinetic movements at the lesioned side. Principle component analysis of DA and DA metabolite levels with dyskinesia scores after l-DOPA/benserazide (6/15 mg/kg) administration indicated a significant correlation only for DA, whereas DA metabolites did not show any significant correlation with the occurrence of dyskinetic behavior. We conclude that intrastriatal l-DOPA itself is not able to induce dyskinetic movements, whereas the increase of intrastriatal DA levels is instrumental for l-DOPA- and DA-induced dyskinetic behavior.