Modeling Parkinson's Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers.

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.

Neuroprotective Effects of the Nutraceutical Dehydrozingerone and Its C2-Symmetric Dimer in a Drosophila Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons responsible for unintended or uncontrollable movements. Mutations in the leucine-rich repeat kinase 2 locus contribute to genetic forms of PD. The fruit fly Drosophila melanogaster carrying this mutation (LRRK2-Dm) is an in vivo model of PD that develops motor impairment and stands for an eligible non-mammalian paradigm to test novel therapeutic approaches. Dehydrozingerone (DHZ) is a natural phenolic compound isolated from ginger and presents anti-inflammatory, antioxidant and neuroprotective properties, making it a potential therapeutic target for PD. We administered DHZ and its C2-symmetric dimer (DHZ-DIM) at 0.5 and 1 mM for 14 and 21 days in the LRRK2-Dm, with the aim of assessing changes in rescuing motor behavior, brain dopaminergic neurons, mitochondria and synapses (T-bars). The shorter treatment with both molecules revealed efficacy at the higher dose, improving climbing behavior with a prevention of dopaminergic neuronal demise. After 21 days, a recovery of the motor disability, dopaminergic neuron loss, mitochondrial damage and T-bars failure was observed with the DHZ-DIM. Our data indicate that the DHZ-DIM exerts a more potent neuroprotective effect with respect to the monomer in LRRK2-Dm, prompting further investigation of these compounds in rodent models of PD.

Combined measure of salivary alpha-synuclein species as diagnostic biomarker for Parkinson's disease.

Parkinson's disease (PD) diagnosis is still vulnerable to bias, and a definitive diagnosis often relies on post-mortem neuropathological diagnosis. In this regard, alpha-synuclein (αsyn)-specific in vivo biomarkers remain a critical unmet need, based on its relevance in the neuropathology. Specifically, content changes in αsyn species such as total (tot-αsyn), oligomeric (o-αsyn), and phosphorylated (p-αsyn) within the cerebrospinal fluid (CSF) and peripheral fluids (i.e., blood and saliva) have been proposed as PD biomarkers possibly reflecting the neuropathological outcome. Here, we measured the p-αsyn levels in the saliva from 15 PD patients along with tot-αsyn, o-αsyn and their ratios, and compared the results with those from 23 healthy subjects (HS), matched per age and sex. We also calculated the optimal cutoff values for different αsyn species to provide information about their capability to discriminate PD from HS. We found that p-αsyn was the most abundant alpha-synuclein species in the saliva. While p-αsyn concentration did not differ between PD and HS when adjusted for total salivary proteins, the ratio p-αsyn/tot-αsyn was largely lower in PD patients than in HS. Moreover, the concentration of o-αsyn was increased in the saliva of PD patients, and tot-αsyn did not differ between PD and HS. The ROC curves indicated that no single αsyn form or ratio could provide an accurate diagnosis of PD. On the other hand, the ratio of different items, namely p-αsyn/tot-αsyn and o-αsyn, yielded more satisfactory diagnostic accuracy, suggesting that the combined measure of different species in the saliva may show more promises as a diagnostic means for PD.

Effects of memantine on mania-like phenotypes exhibited by Drosophila Shaker mutants.

INTRODUCTION: Increased glutamate levels and electrolytic fluctuations have been observed in acutely manic patients. Despite some efficacy of the non-competitive NMDA receptor antagonist memantine (Mem), such as antidepressant-like and mood-stabilizer drugs in clinical studies, its specific mechanisms of action are still uncertain. The present study aims to better characterize the Drosophila melanogaster fly Shaker mutants (SH), as a translational model of manic episodes within bipolar disorder in humans, and to investigate the potential anti-manic properties of Mem. METHODS AND RESULTS: Our findings showed typical behavioral abnormalities in SH, which mirrored with the overexpression of NMDAR-NR1 protein subunit, matched well to glutamate up-regulation. Such molecular features were associated to a significant reduction of SH brain volume in comparison to Wild Type strain flies (WT). Here we report on the ability of Mem treatment to ameliorate behavioral aberrations of SH (similar to that of Lithium), and its ability to reduce NMDAR-NR1 over-expression. CONCLUSIONS: Our results show the involvement of the glutamatergic system in the SH, given the interaction between the Shaker channel and the NMDA receptor, suggesting this model as a promising tool for studying the neurobiology of bipolar disorders. Moreover, our results show Mem as a potential disease-modifying therapy, providing insight on new mechanisms of action.

Improvements of Motor Performances in the Drosophila LRRK2 Loss-of-Function Model of Parkinson's Disease: Effects of Dialyzed Leucocyte Extracts from Human Serum.

Within neurodegenerative syndromes, Parkinson's disease (PD) is typically associated with its locomotor defects, sleep disturbances and related dopaminergic (DA) neuron loss. The fruit fly, Drosophila melanogaster (D. melanogaster), with leucine-rich repeat kinase 2 mutants (LRRK2) loss-of-function in the WD40 domain, provides mechanistic insights into corresponding human behaviour, possibly disclosing some physiopathologic features of PD in both genetic and sporadic forms. Moreover, several data support the boosting impact of innate and adaptive immunity pathways for driving the progression of PD. In this context, human dialyzable leukocyte extracts (DLE) have been extensively used to transfer antigen-specific information that influences the activity of various immune components, including inflammatory cytokines. Hence, the main goal of our study was to ascertain the therapeutic potential of DLE from male and female donors on D. melanogaster LRRK2 loss-of-function, as compared to D. melanogaster wild-type (WT), in terms of rescuing physiological parameters, such as motor and climbing activities, which are severely compromised in the mutant flies. Finally, in search of the anatomical structures responsible for restored functions in parkinsonian-like mutant flies, we found a topographical correlation between improvement of locomotor performances and an increased number of dopaminergic neurons in selective areas of LRRK2 mutant brains.

Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2WD40 Genetic Model of Parkinson's Disease.

The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2WD40), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. Here the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2WD40 genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2WD40 flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2WD40 fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2WD40 flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans.

Rimonabant, a potent CB1 cannabinoid receptor antagonist, is a Gαi/o protein inhibitor.

Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gαi/o-type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [35S]GTPγS binding to cortical membranes of CB1- and GABAB-receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABAB or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABAB receptors. Rimonabant prevented G protein-mediated GABAB and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293 cells and to G protein-coupled inwardly rectifying K+ channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABAB receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABAB receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gαi/o-type G proteins. The inhibition of Gαi/o protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations.

Central effects of 1,4-butanediol are mediated by GABA(B) receptors via its conversion into gamma-hydroxybutyric acid.

The aliphatic alcohol 1,4-butanediol in converted into gamma-hydroxybutyric acid (GHB) via two enzymatic steps: first, it is oxidised by alcohol dehydrogenase in gamma-hydroxybutyraldehyde; second, the latter is transformed, likely by aldehyde dehydrogenase, into GHB. Initially, the present study compared the sedative/hypnotic effect of GHB and 1,4-butanediol, measured as loss of righting reflex. 1,4-Butanediol was more potent than GHB, presumably because of a more rapid penetration of the blood brain barrier. Further alcohol dehydrogenase inhibitors, 4-methylpyrazole and ethanol, totally prevented the sedative/hypnotic effect of 1,4-butanediol; the aldehyde dehydrogenase inhibitor disulfiram partially blocked the sedative/hypnotic effect of 1,4-butanediol. Finally, the sedative/hypnotic effect of 1,4-butanediol was antagonised by the GABA(B) receptor antagonists, SCH 50911 [(2S)(+)-5,5-dimethyl-2-morpholineacetic acid] and CGP 46381 [(3-aminopropyl)(cyclohexylmethyl)phosphinic acid], but not by the putative GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid), indicating that it is mediated by GABA(B) but not GHB receptors. Taken together, these results suggest that the sedative/hypnotic effect of 1,4-butanediol is mediated by its conversion in vivo into GHB which, in turn, binds to GABA(B) receptors. Accordingly 1,4-butanediol, unlike GHB, failed to displace [(3)H]GHB and [(3)H]baclofen in brain membranes.

Stereoselectivity of NCS-382 binding to gamma-hydroxybutyrate receptor in the rat brain.

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of gamma-aminobutyric acid (GABA), has been postulated to act both as a specific agonist of GHB receptors and as a weak GABA(B) receptor agonist. The racemic compound 6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid (RS-NCS-382), the only available antagonist of GHB receptors, has been resolved in two enantiomers, R- and S-; the potency of the latter to displace 4-hydroxy [2-3-(3)H] butyric acid ([(3)H]GHB) and [(3)H]NCS-382 from GHB receptors, on one hand, and [(3)H]baclofen from GABA(B) receptors on the other was compared in rat brain homogenates. R-NCS-382 was found to be twice and 60 times more potent than the RS- and S-forms, respectively, in displacing [(3)H]GHB and 2 and 14 times, respectively, in displacing [(3)H]NCS-382 from GHB binding. Neither RS-NCS-382 nor its enantiomers inhibited [(3)H]baclofen binding up to a concentration of 1 mM. Our results demonstrate that R-NCS-382 is the enantiomer of RS-NCS-382 with higher affinity for GHB receptors.

Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid.

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.