Ruthenium p-Cymene Complexes Incorporating Substituted Pyridine-Quinoline-Based Ligands: Synthesis, Characterization, and Cytotoxic Properties.

Organometallic complexes of the formula [Ru(N^N)(p-cymene)Cl][X] (N^N = bidentate polypyridyl ligands, p-cymene = 1-methyl-4-(1-methylethyl)-benzene, X = counter anion), are currently studied as possible candidates for the potential treatment of cancer. Searching for new organometallic compounds with good to moderate cytotoxic activities, a series of mononuclear water-soluble ruthenium(II)-arene complexes incorporating substituted pyridine-quinoline ligands, with pending -CH2OH, -CO2H and -CO2Me groups in the 4-position of quinoline ring, were synthesized, for the first time, to study their possible effect to modulate the activity of the ruthenium p-cymene complexes. These include the [Ru(η6-p-cymene)(pqhyme)Cl][X] (X = Cl- (1-Cl), PF6- (1-PF6), pqhyme = 4-hydroxymethyl-2-(pyridin-2-yl)quinoline), [Ru(η6-p-cymene)(pqca)Cl][Cl] ((2-Cl), pqca = 4-carboxy-2-(pyridin-2-yl)quinoline), and [Ru(η6-p-cymene)(pqcame)Cl][X] (X = Cl- (3-Cl), PF6- (3-PF6), pqcame = 4-carboxymethyl-2-(pyridin-2-yl)quinoline) complexes, respectively. Identification of the complexes was based on multinuclear NMR and ATR-IR spectroscopic methods, elemental analysis, conductivity measurements, UV-Vis spectroscopic, and ESI-HRMS techniques. The solid-state structures of 1-PF6 and 3-PF6 have been elucidated by single-crystal X-ray diffraction revealing a three-legged piano stool geometry. This is the first time that the in vitro cytotoxic activities of these complexes are studied. These were conducted in HEK293T (human embryonic kidney cells) and HeLa cells (cervical cancer cells) via the MTT assay. The results show poor in vitro anticancer activities for the HeLa cancer cell lines and 3-Cl proved to be the most potent (IC50 > 80 µΜ). In both cell lines, the cytotoxicity of the ligand precursor pqhyme is significantly higher than that of cisplatin.

First-Row Transition Metal Complexes Incorporating the 2-(2'-pyridyl)quinoxaline Ligand (pqx), as Potent Inflammatory Mediators: Cytotoxic Properties and Biological Activities against the Platelet-Activating Factor (PAF) and Thrombin.

Inflammatory mediators constitute a recently coined term in the field of metal-based complexes with antiplatelet activities. Our strategy targets Platelet-Activating Factor (PAF) and its receptor, which is the most potent lipid mediator of inflammation. Thus, the antiplatelet (anti-PAF) potency of any substance could be exerted by inhibiting the PAF-induced aggregation in washed rabbit platelets (WRPs), which internationally is a well-accepted methodology. Herein, a series of mononuclear (mer-[Cr(pqx)Cl3(H2O]) (1), [Co(pqx)Cl2(DMF)] (2) (DMF = N,N'-dimethyl formamide), [Cu(pqx)Cl2(DMSO)] (3) (DMSO = dimethyl sulfoxide), [Zn(pqx)Cl2] (4)) and dinuclear complexes ([Mn(pqx)(H2O)2Cl2]2 (5), [Fe(pqx)Cl2]2 (6) and [Ni(pqx)Cl2]2 (7)) incorporating the 2-(2'-pyridyl)quinoxaline ligand (pqx), were biologically evaluated as inhibitors of the PAF- and thrombin-induced aggregation in washed rabbit platelets (WRPs). The molecular structure of the five-co-ordinate analog (3) has been elucidated by single-crystal X-ray diffraction revealing a trigonal bipyramidal geometry. All complexes are potent inhibitors of the PAF-induced aggregation in WRPs in the micromolar range. Complex (6) displayed a remarkable in vitro dual inhibition against PAF and thrombin, with IC50 values of 1.79 µM and 0.46 µM, respectively. Within the series, complex (5) was less effective (IC50 = 39 µM) while complex (1) was almost 12-fold more potent against PAF, as opposed to thrombin-induced aggregation. The biological behavior of complexes 1, 6 and 7 on PAF's basic metabolic enzymatic pathways reveals that they affect key biosynthetic and catabolic enzymes of PAF underlying the anti-inflammatory properties of the relevant complexes. The in vitro cytotoxic activities of all complexes in HEK293T (human embryonic kidney cells) and HeLa cells (cervical cancer cells) are described via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results reveal that complex 3 is the most potent within the series.

Deficiency of the serine peptidase Kallikrein 6 does not affect the levels and the pathological accumulation of a-synuclein in mouse brain.

Several lines of evidence indicate that the propagation of misfolded α-synuclein (α-syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α-syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α-syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α-syn degrading enzymes and has been implicated in the clearance of extracellular α-syn. However, the physiological role of this enzyme in regulating α-syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock-out (Klk6-/- ) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α-syn processing. Behavioral phenotyping showed that Klk6-/- mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α-syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α-syn. Upon in vivo administration of α-syn pre-formed fibrils (PFF), α-syn pathologic accumulations were evident both in the brains of Klk6-/- mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α-syn levels observed in the A53T α-syn over-expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α-syn fibrils in α-syn turnover studies.

Changes in the cellular fatty acid profile drive the proteasomal degradation of α-synuclein and enhance neuronal survival.

Parkinson's disease is biochemically characterized by the deposition of aberrant aggregated α-synuclein in the affected neurons. The aggregation properties of α-synuclein greatly depend on its affinity to bind cellular membranes via a dynamic interaction with specific lipid moieties. In particular, α-synuclein can interact with arachidonic acid (AA), a polyunsaturated fatty acid, in a manner that promotes the formation of α-helix enriched assemblies. In a cellular context, AA is released from membrane phospholipids by phospholipase A2 (PLA2 ). To investigate the impact of PLA2 activity on α-synuclein aggregation, we have applied selective PLA2 inhibitors to a SH-SY5Y cellular model where the expression of human wild-type α-synuclein is correlated with a gradual accumulation of soluble oligomers and subsequent cell death. We have found that pharmacological and genetic inhibition of GIVA cPLA2 resulted in a dramatic decrease of intracellular oligomeric and monomeric α-synuclein significantly promoting cell survival. Our data suggest that alterations in the levels of free fatty acids, and especially AA and adrenic acid, promote the formation of α-synuclein conformers which are more susceptible to proteasomal degradation. This mechanism is active only in living cells and is generic since it does not depend on the absolute quantity of α-synuclein, the presence of disease-linked point mutations, the expression system or the type of cells. Our findings indicate that the α-synuclein-fatty acid interaction can be a critical determinant of the conformation and fate of α-synuclein in the cell interior and, as such, cPLA2 inhibitors could serve to alleviate the intracellular, potentially pathological, α-synuclein burden.

Modulation of ß-glucocerebrosidase increases α-synuclein secretion and exosome release in mouse models of Parkinson's disease.

Glucocerebrosidase gene (GBA) mutations are the most common genetic contributor to Parkinson's disease (PD) and are associated with decreased glucocerebrosidase (GCase) enzymatic activity in PD. PD patients without GBA mutations also exhibit lower levels of GCase activity in the central nervous system suggesting a potential contribution of the enzyme activity in disease pathogenesis, possibly by alteration of lysosomal function. α-synuclein (ASYN), a protein with a central role in PD pathogenesis, has been shown to be secreted partly in association with exosomes. It is possible that a dysfunction of the endocytic pathway through GCase may result in altered exosome release of ASYN. The aim of this study was to examine whether manipulating GCase activity in vivo and in vitro could affect ASYN accumulation and secretion. GCase overexpression in vitro resulted in a significant decrease of exosome secretion. Chronic inhibition of GCase activity in vivo, by administration of the covalent inhibitor conduritol-B epoxide in A53T-synuclein alpha gene Tg mice significantly elevated intracellular oligomeric ASYN species. Importantly, GCase inhibition, induced a profound increase in the number of brain exosomes released, as well as exosome-associated ASYN oligomers. Finally, virus-mediated expression of mutant GBA in the mouse striatum increased ASYN secretion in the same region. Together, these results provide for the first time evidence that a decrease of GCase or overexpression of mutant GCase in a chronic in vivo setting can affect ASYN secretion. Such effects may mediate enhanced propagation of ASYN, driving pathology in GBA-associated PD.

High discriminatory ability of peripheral and CFSF biomarkers in Lewy body diseases.

Differential diagnosis between Parkinson's disease (PD) Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), namely spectrum of Lewy bodies disorders (LBDs), may be challenging, and their common underlying pathophysiology is debated. Our aim was to examine relationships among neurodegenerative biomarkers [alpha-synuclein (α-Syn), Alzheimer's Disease (AD)-related (beta-amyloid Aß42, tau [total τΤ and phosphorylated τp-181]), dopaminergic imaging (DATSCAN-SPECT)] and spectrum of LBD. This is a cross-sectional prospective study in 30 PD, 18 PDD, 29 DLB patients and 30 healthy controls. We compared α-Syn in CSF, plasma and serum and CSF Aß42, τΤ and τp-181 across these groups. Correlations between such biomarkers and motor, cognitive/neuropsychiatric tests, and striatal asymmetry indexes were examined. CSF α-Syn was higher in DLB versus PD/PDD/controls, and lower in PD and PDD patients compared to controls (all p < 0.001). Serum α-Syn levels were higher in all patient groups compared to controls. After excluding those DLB patients with CSF AD profile, plasma and serum Syn levels were higher in the LBD group as a whole compared to controls. The combination of CSF α-Syn, serum α-Syn and Aß42 for comparison between PD and DLB [AUC = 0.96 (95% CI 0.90-1.00)] was significantly better when compared to serum α-Syn alone (p < 0.001). Correlation analyses of biomarkers with cognitive/neuropsychiatric scales revealed some associations, but no consistent, cohesive picture. Peripheral biomarkers such as serum α-Syn, and CSF α-Syn and Aß42 may contribute as potential biomarkers to separate LBDs from controls and to differentiate DLB from the other LBDs with high sensitivity and specificity among study groups.

How is alpha-synuclein cleared from the cell?

The levels and conformers of alpha-synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha-synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha-synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha-synuclein species, in a manner that depends on alpha-synuclein post-translational modifications. Extracellular alpha-synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha-synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. This article is part of the Special Issue "Synuclein".

Plasma alpha-synuclein levels in patients with Parkinson's disease: a systematic review and meta-analysis.

OBJECTIVE: To date, there are no definitive biomarkers for diagnose Parkinson's disease (PD). The detection of α-synuclein (α-Syn) in plasma of PD patients has yielded promising but inconclusive results. To determine the performance of α-Syn as a diagnostic biomarker of PD, we used a meta-analysis. METHODS: We identified 173 studies through a systematic literature review. From those, only studies reporting data on total α-Syn levels were included in the meta-analysis (10 publications, 1302 participants). Quality of studies was assessed by Newcastle-Ottawa scale. RESULTS: The α-Syn levels were significantly higher in PD patients than healthy controls (standardized mean difference [SMD] = 0.778, 95% confidence interval = 0.284 to 1.272, p = 0.002). Similar results were found after omitting any individual study from meta-analysis, with SMD ranges from 0.318 (95% CI = 0.064 to 0.572, p = 0.014) to 0.914 (95% CI = 0.349 to 1.480, p = 0.002). According to meta-regression analysis, increased mean patients age (slope = - 0.232, 95% CI = - 0.456 to - 0.008, p = 0.042), increased total number of participants (slope = - 0.007, 95% CI = - 0.013 to - 0.0004, p = 0.038), and increased percentage of males (slope = - 6.444, 95% CI = - 10.841 to - 2.047, p = 0.004) were associated with decreased SMD of α-Syn levels across studies. We did not find any significant association between the SMD in α-Syn levels and disease duration, disease severity, and quality of studies. Most of studies applied ELISA assays. CONCLUSION: Total plasma α-Syn levels were higher in PD patients than controls. Analytical factors were important limitations.

GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum.

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease.

The small GTPase Rab11 co-localizes with α-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity.

Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.