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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.
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Antineoplásicos , Complexos de Coordenação , Cimenos , Piridinas , Quinolinas , Rutênio , Humanos , Rutênio/química , Quinolinas/química , Quinolinas/síntese química , Quinolinas/farmacologia , Ligantes , Cimenos/química , Cimenos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Piridinas/química , Piridinas/síntese química , Piridinas/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Estrutura Molecular , Linhagem Celular Tumoral , Cristalografia por Raios X , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: It is now realized that Parkinson's disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved. METHODS: To investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro. RESULTS: We found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential. CONCLUSIONS: Our work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation.
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Doença de Parkinson , alfa-Sinucleína , Humanos , Camundongos , Animais , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , NF-kappa B/metabolismo , Astrócitos/metabolismo , Doenças Neuroinflamatórias , Sinalização do Cálcio , Doença de Parkinson/metabolismo , Camundongos Transgênicos , CitocinasRESUMO
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.
Assuntos
Antineoplásicos , Complexos de Coordenação , Elementos de Transição , Animais , Humanos , Coelhos , Agregação Plaquetária , Fator de Ativação de Plaquetas/farmacologia , Fator de Ativação de Plaquetas/metabolismo , Plaquetas/metabolismo , Trombina/metabolismo , Complexos de Coordenação/farmacologia , Complexos de Coordenação/metabolismo , Ligantes , Mediadores da Inflamação/metabolismo , Dimetil Sulfóxido/farmacologia , Quinoxalinas/farmacologia , Células HEK293 , Células HeLa , Antineoplásicos/farmacologia , Elementos de Transição/metabolismoRESUMO
Even though it is currently well-established that α-synuclein aggregation is closely associated with the pathological events in Parkinson's disease (PD) and several other neurodegenerative disorders, collectively called synucleinopathies, the mechanistic link between α-synuclein aggregates and the onset and progression of neurodegeneration in these diseases remain unclear. The process of aggregation initiates from a structurally distorted monomer that gradually oligomerizes to generate a repertoire of fibrillar and oligomeric multimers that deposit within diseased cells in the brain. Total α-synuclein has been proposed as a potential biomarker in PD, but most of the studies do not discriminate between distinct α-synuclein conformers. To correlate protein measurements to disease pathology, we have developed a conformation-specific ELISA method that selectively detects fibrillar and oligomeric forms of α-synuclein without cross-reacting with monomers. We have used this assay to determine the levels of aggregated α-synuclein in human and mouse brain tissue as well as in CSF and CSF-derived exosomes from patients with synucleinopathy and control subjects. Our results verify the ability of the new assay to detect aggregated α-synuclein in complex matrices and support the idea that the levels of these conformers are related to the age of onset in PD patients, while CSF analysis showed that these species exist in low abundance in CSF and CSF-derived exosomes. Future studies will be required to fully assess the diagnostic usefulness of this ELISA in synucleinopathies.
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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.
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Encéfalo/metabolismo , Encéfalo/patologia , Calicreínas/deficiência , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Feminino , Calicreínas/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinucleinopatias/genética , alfa-Sinucleína/genéticaRESUMO
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.
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Ácido Araquidônico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Neurônios/citologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/química , alfa-Sinucleína/metabolismo , Sobrevivência Celular , Células Cultivadas , Humanos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , ProteóliseRESUMO
α-Synuclein (α-syn) has been genetically and biochemically linked to the pathogenesis of Parkinson's disease (PD). There is accumulating evidence that misfolded α-syn species spread between cells in a prion-like manner and seed the aggregation of endogenous protein in the recipient cells. Exosomes have been proposed to mediate the transfer of misfolded α-syn and thus facilitate disease transmission, although the pathological mechanism remains elusive. Here, we investigated the seeding capacity of exosome-associated α-syn, in vivo. Disease-associated α-syn was present in exosome fractions isolated from transgenic A53T mouse brain. However, following intrastriatal injection of such exosomes in wild-type (wt) mice, we were not able to detect any accumulation of endogenous α-syn. In addition, recombinant fibrillar α-syn, when loaded to isolated brain exosomes, induced minor pathological α-syn brain accumulation at 7 months post injection. These data suggest that exosomes neutralize the effect of toxic α-syn species and raise additional questions on their paracrine modulatory role in disease transmission.
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BACKGROUND: Variations of α-synuclein levels have been reported in serum and plasma in Parkinson's Disease (PD) Patients. METHODS: Serum and plasma were obtained from PD patients without known mutations (GU-PD, n = 124)), carriers of the A53T/G209A point mutation in the α-synuclein gene (SNCA) (n = 29), and respective age-/sex-matched controls. Levels of total α-synuclein were assessed using an in-house ELISA assay. RESULTS: A statistically significant increase of α-synuclein levels was found in serum, but not plasma, from GU-PD patients compared to healthy controls. A statistically significant decrease of α-synuclein levels was found in serum and plasma from symptomatic A53T mutation carriers compared to healthy controls. Plasma α-synuclein levels were modestly negatively correlated with UPDRS part III score and disease duration in A53T-PD patients. CONCLUSION: Increased α-synuclein levels in serum of GU-PD patients suggest a systemic deregulation of α-synuclein homeostasis in PD. The opposite results in A53T-PD highlight the complexity of α-synuclein homeostatic regulation in PD, and suggest the possibility of reduced expression of the mutant allele.
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Doença de Parkinson/sangue , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , alfa-Sinucleína/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , alfa-Sinucleína/genéticaRESUMO
Recent evidence suggests a potential role for mixed proteinopathies in the development of clinical manifestations in patients with Huntington's disease (HD). A possible cross-talk between mutant huntingtin and α-synuclein aggregates has been postulated. Serum α-synuclein has been evaluated as a potential biomarker in patients with Parkinson's disease (PD). We presently sought to investigate serum α-synuclein levels in 38 HD patients (34 symptomatic and 4 premanifest) and compare them to 36 controls. We found that α-synuclein was elevated in HD patients vs. controls (2.49⯱â¯1.47 vs. 1.40⯱â¯1.16, pâ¯=â¯0.001). There was no difference in α-synuclein levels between symptomatic vs. premanifest HD, nor between HD patients receiving medication vs. treatment-naïve. Furthermore, α-synuclein levels showed no correlation with CAG2, Unified HD Rating Scale (UHDRS) motor score, age, disease duration or disease burden score. Our results provide evidence for elevated serum α-synuclein in HD and lend support to further investigating the role of α-synuclein in this disorder.
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Doença de Huntington , Doença de Parkinson , Humanos , alfa-SinucleínaRESUMO
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.
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Peptídeos beta-Amiloides/metabolismo , Demência/diagnóstico , Demência/metabolismo , Doença de Parkinson/diagnóstico , Doença de Parkinson/metabolismo , Fragmentos de Peptídeos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Idoso , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores , Demência/diagnóstico por imagem , Demência/fisiopatologia , Diagnóstico Diferencial , Feminino , Humanos , Doença por Corpos de Lewy/diagnóstico , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/fisiopatologia , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/fisiopatologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Tomografia Computadorizada de Emissão de Fóton Único , alfa-Sinucleína/sangue , alfa-Sinucleína/líquido cefalorraquidiano , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidianoRESUMO
Alpha-synuclein (α-syn) is biochemically and genetically linked to Parkinson's disease (PD) and other synucleinopathies. It is now widely accepted that α-syn can be released in the extracellular space, even though the mechanism of its release is still unclear. In addition, pathology-related aggregated species of α-syn have been shown to propagate between neurons in synaptically connected areas of the brain thereby assisting the spreading of pathology in healthy neighboring neuronal cells. In neurons, calcium channels are key signaling elements that modulate the release of bioactive molecules (hormones, proteins, and neurotransmitters) through calcium sensing. Such calcium sensing activity is determined by the distinct biophysical and pharmacological properties and the ability of calcium channels to interact with other modulatory proteins. Although the function of extracellular α-syn is currently unknown, previous work suggested the presence of a calcium-dependent mechanism for α-syn secretion both in vitro, in neuronal cells in culture, and also in vivo, in the context of a trans-neuronal network in brain. Mechanisms regulating extracellular α-syn levels may be of particular importance as they could represent novel therapeutic targets. We discuss here how calcium channel activity may contribute to α-syn aggregation and secretion as a pathway to disease progression in synucleinopathies.
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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".
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alfa-Sinucleína/metabolismo , Astrócitos/metabolismo , Progressão da Doença , Exossomos/metabolismo , Líquido Extracelular/metabolismo , Terapia Genética , Humanos , Imunoterapia , Líquido Intracelular/metabolismo , Corpos de Lewy/metabolismo , Lisossomos/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregação Patológica de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ubiquitinação , alfa-Sinucleína/químicaRESUMO
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.
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Doença de Parkinson/sangue , alfa-Sinucleína/sangue , Biomarcadores/sangue , HumanosRESUMO
INTRODUCTION: Given the overlapping of neuropathological, neurochemical and neuropsychiatric profiles of Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), their differential diagnosis is challenging. Specific neuropsychiatric features or biomarkers, such as cerebrospinal fluid (CSF) α-Synuclein (α-Syn), may aid in differential diagnosis. This study aims to compare the neuropsychiatric and CSF α-Syn profiles in these conditions, and to investigate the possible association between CSF α-Syn levels and neuropsychiatric symptoms. METHODS: We conducted a prospective cross-sectional study, between January 2013 and January 2015, with 16 PDD, 28 DLB and 19 AD patients. All participants underwent a detailed clinical, neuropsychological, neuropsychiatric [Neuropsychiatric Inventory (NPI)] and CSF α-Syn analysis. RESULTS: Significantly greater NPI Hallucinations Subitem score was found in the PDD and DLB groups compared to AD (both p < 0.001). NPI Agitation score was greater in the DLB compared to PDD group (p = 0.012). NPI Sleep score was greater in the DLB compared to AD group (p = 0.001). Total NPI score was greater in the DLB compared to AD and PDD groups. To discriminate between the DLB and AD and between DLB and PDD groups, logistic regression analysis showed that both NPI scores and α-Syn levels were independently associated. There was no correlation between NPI scores and α-Syn levels. Increased NPI scores and α-Syn levels are associated with greater likelihood for being in DLB than in PDD or AD groups. ROC analysis showed that the combination of NPI and α-Syn increases the discriminative ability of each marker alone (p < 0.001) with AUC equal to 0.95 (95% CI 0.91-0.99). CONCLUSION: NPI scores and CSF α-Syn levels were useful as independent variables to differentiate DLB from PDD and AD.
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Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/psicologia , Doença por Corpos de Lewy/líquido cefalorraquidiano , Doença por Corpos de Lewy/psicologia , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/psicologia , Idoso , Biomarcadores/líquido cefalorraquidiano , Estudos Transversais , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Estudos Prospectivos , alfa-Sinucleína/líquido cefalorraquidianoRESUMO
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.
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Exossomos/genética , Glucosilceramidase/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Exossomos/metabolismo , Glucosilceramidase/metabolismo , Humanos , Inositol/administração & dosagem , Inositol/análogos & derivados , Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Mutação , Doença de Parkinson/fisiopatologiaRESUMO
The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct "autophagosome-exosome-like" profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.
Assuntos
Autofagossomos/metabolismo , Autofagia/fisiologia , Exossomos/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Células Cultivadas , Cloroquina/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Humanos , Doença por Corpos de Lewy/metabolismo , Lisossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Doença de Parkinson/metabolismo , Transporte Proteico , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Differential diagnosis of Parkinson-plus patients (PSP, CBD, MSA) and Parkinson's disease (PD) patients is often not straightforward, particularly in atypical cases or at the initial stages of the diseases. Classic CSF biomarkers (amyloid-beta - Aß42, tau protein - τT and phosphorylated tau protein - τP-181) are established biomarkers in the diagnosis of Alzheimer's disease (AD). CSF a-synuclein (α-syn) has emerged as a promising biomarker in patients with Parkinsonism. The aim of this study was to analyze the CSF biochemical profile of patients with Parkinsonism. METHODS: We analyzed the CSF biomarker profile (Aß42, τT, τP-181, α-syn) and all relevant ratios in 68 patients with Parkinsonism (19 PSP, 15 MSA, 17 CBD, 17 PD) and 18 controls, diagnosed by latest established diagnostic criteria. RESULTS: CBD patients exhibited elevated τT and decreased Aß42 compared to the other groups. Five CBD, one PSP patient and one control had a typical AD CSF profile. After exclusion of these patients, the τT/Aß42 ratio was significantly elevated in MSA patients compared to PD patients and provided excellent specificity and adequate sensitivity in their differential diagnosis. CONCLUSION: CSF biochemical profile analysis is important in distinguishing AD patients with a CBS phenotype from non-AD CBS patients. The τT/Aß42 ratio is useful in the differential diagnosis of MSA from PD.
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Atrofia de Múltiplos Sistemas/líquido cefalorraquidiano , Doença de Parkinson/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Paralisia Supranuclear Progressiva/líquido cefalorraquidiano , alfa-Sinucleína/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Idoso , Biomarcadores/líquido cefalorraquidiano , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Curva ROC , Sistema de RegistrosRESUMO
It is now established that α-synuclein can be physiologically secreted to the extracellular space. In this sense, mechanisms that govern the secretion of the protein may be of importance in the initiation and progress of synucleinopathies. It is possible that increased secretion may aid the formation of toxic seeds extracellularly. Alternatively, reduced presence of extracellular α-synuclein due to impaired secretion may increase the intracellular load and trigger intracellular seeding. Once outside, α-synuclein can exert various paracrine actions on neighboring cells again by mechanisms that have not been fully elucidated. It has been demonstrated that, when applied extracellularly, α-synuclein species can induce multiple neurotoxic and inflammatory responses, and aid the transmission of pathology between neurons. Still, the exact mechanism(s) by which secreted α-synuclein affects the homeostasis of other neurons is still not well understood. A portion of α-synuclein has been shown to be associated with the surface and lumen of exosomes which can transfer it to the surrounding cells, and potentially trigger seeding. Interestingly, increased exosome release has been linked to pathological situations of lysosomal dysfunction as observed in Parkinson's disease (PD). However, the possibility that the observed α-synuclein pathology spread is attributable to the passive diffusion of the initial injected α-synuclein strains cannot be excluded. Importantly, most of the studies that have so far addressed the role of extracellular α-synuclein have not employed naturally secreted forms of the protein. It is plausible that deregulation in the normal processing of secreted α-synuclein may aid the formation of "toxic" species and as such it may also be a causative risk factor for PD. In this capacity, elucidation of the underlying mechanisms that regulate the protein-levels of extracellular α-synuclein becomes essential. Such mechanisms could involve its proteolytic clearance from the extracellular milieu.
Assuntos
Doenças Neurodegenerativas , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Exocitose , HumanosRESUMO
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.
Assuntos
Corpo Estriado/metabolismo , Canais KATP/metabolismo , Transmissão Sináptica/fisiologia , alfa-Sinucleína/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Moduladores GABAérgicos/farmacologia , Humanos , Canais KATP/agonistas , Canais KATP/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de GABA-B/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Cerebrospinal fluid (CSF) α-synuclein (ASYN) levels are emerging as a possible biomarker in a number of neurodegenerative conditions; however, there has been little study of such levels in demyelinating conditions with neurodegeneration such as multiple sclerosis (MS). In this study, we aimed to assess CSF ASYN levels in MS spectrum [clinically isolated syndrome (CIS) and MS] patients and compare them to those obtained in control subjects with benign neurological conditions (BNC). We used a recently developed, ultra-sensitive sandwich enzyme-linked immunosorbent assay to measure and compare CSF ASYN levels in three categories of subjects: BNC (n = 38), CIS (n = 36) and MS [Relapsing Remitting (RRMS, n = 22) and Primary Progressive (PPMS, n = 15)]. We also performed secondary analyses, including relationship of CSF ASYN levels to aging, gender, presence of CSF oligoclonal bands (OB) and gadolinium (Gd)-enhancing demyelinating lesions on T1-weighted MRIs. CSF ASYN levels were found to be significantly lower in the CIS (78.2 ± 7.5 pg/mL), RRMS (76.8 ± 5.1 pg/mL), and PPMS (76.3 ± 6.7 pg/mL) groups compared to the BNC (125.7 ± 13.6 pg/mL) group. Secondary analyses did not reveal additional correlations. Our results suggest that in a cohort of CIS and MS patients, CSF ASYN levels are decreased, thus providing another possible link between MS and neurodegeneration. Future studies will need to be performed to confirm and extend these findings, to lead to a fuller understanding of the possible biological link between ASYN and MS. Alpha-synuclein levels in the Cerebrosinal Fluid (CSF) may reflect neurodegenerative processes. Here we measure CSF alpha-synuclein in demyelinating conditions ranging from Clinically Isolated Syndrome to Primary Progressive Multiple Sclerosis (MS). We find a similar magnitude of decreased alpha-synuclein compared to a control group in all such MS spectrum conditions; such a decrease may reflect an underlying early neurodegenerative disease process.