Neurodegeneration, oxidative stress and lipid metabolism plasma biomarkers to differentiate Parkinson's disease from atypical parkinsonian syndromes.

INTRODUCTION: The identification of blood biomarkers appears to be a means of improving diagnosis accuracy in Parkinson's disease (PD) and atypical parkinsonian syndromes (APS). We, therefore, evaluate the performance of neurodegeneration, oxidative stress and lipid metabolism plasma biomarkers to distinguish PD from APS. METHODS: This was a monocentric study with a cross-sectional design. Plasma levels and discriminating power of neurofilament light chain (NFL), malondialdehyde (MDA) and 24S-Hydroxycholesterol (24S-HC) were assessed in patients with clinical diagnoses of PD or APS. RESULTS: In total, 32 PD cases and 15 APS cases were included. Mean disease durations were 4.75 years in PD group and 4.2 years in APS group. Plasma levels of NFL, MDA and 24S-HC differed significantly between the APS and PD groups (P=0.003; P=0.009; P=0.032, respectively). NFL, MDA and 24S-HC discriminated between PD and APS (AUC=0.76688; AUC=0.7375; AUC=0.6958, respectively). APS diagnosis significantly increased with MDA level≥23.628nmol/mL (OR: 8.67, P=0.001), NFL level≥47.2pg/mL (OR: 11.92, P<0.001) or 24S-HC level≤33.4pmol/mL (OR: 6.17, P=0.008). APS diagnosis considerably increased with the combination of NFL and MDA levels beyond cutoff values (OR: 30.67, P<0.001). Finally, the combination of NFL and 24S-HC levels, or MDA and 24S-HC levels, or all three biomarkers' levels beyond cutoff values systematically classified patients in the APS group. CONCLUSION: Our results suggest that 24S-HC and especially MDA and NFL could be helpful for differentiating PD from APS. Further studies will be needed to reproduce our findings on larger, prospective cohorts of patients with parkinsonism evolving for less than 3 years.

Attenuation of 7-ketocholesterol- and 7ß-hydroxycholesterol-induced oxiapoptophagy by nutrients, synthetic molecules and oils: Potential for the prevention of age-related diseases.

Age-related diseases for which there are no effective treatments include cardiovascular diseases; neurodegenerative diseases such as Alzheimer's disease; eye disorders such as cataract and age-related macular degeneration; and, more recently, Severe Acute Respiratory Syndrome (SARS-CoV-2). These diseases are associated with plasma and/or tissue increases in cholesterol derivatives mainly formed by auto-oxidation: 7-ketocholesterol, also known as 7-oxo-cholesterol, and 7ß-hydroxycholesterol. The formation of these oxysterols can be considered as a consequence of mitochondrial and peroxisomal dysfunction, leading to increased in oxidative stress, which is accentuated with age. 7-ketocholesterol and 7ß-hydroxycholesterol cause a specific form of cytotoxic activity defined as oxiapoptophagy, including oxidative stress and induction of death by apoptosis associated with autophagic criteria. Oxiaptophagy is associated with organelle dysfunction and in particular with mitochondrial and peroxisomal alterations involved in the induction of cell death and in the rupture of redox balance. As the criteria characterizing 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cytotoxicity are often simultaneously observed in major age-related diseases (cardiovascular diseases, age-related macular degeneration, Alzheimer's disease) the involvement of these oxysterols in the pathophysiology of the latter seems increasingly likely. It is therefore important to better understand the signalling pathways associated with the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol in order to identify pharmacological targets, nutrients and synthetic molecules attenuating or inhibiting the cytotoxic activities of these oxysterols. Numerous natural cytoprotective compounds have been identified: vitamins, fatty acids, polyphenols, terpenes, vegetal pigments, antioxidants, mixtures of compounds (oils, plant extracts) and bacterial enzymes. However, few synthetic molecules are able to prevent 7-ketocholesterol- and/or 7ß-hydroxycholesterol-induced cytotoxicity: dimethyl fumarate, monomethyl fumarate, the tyrosine kinase inhibitor AG126, memantine, simvastatine, Trolox, dimethylsufoxide, mangafodipir and mitochondrial permeability transition pore (MPTP) inhibitors. The effectiveness of these compounds, several of which are already in use in humans, makes it possible to consider using them for the treatment of certain age-related diseases associated with increased plasma and/or tissue levels of 7-ketocholesterol and/or 7ß-hydroxycholesterol.

Induction of a non-apoptotic mode of cell death associated with autophagic characteristics with steroidal maleic anhydrides and 7ß-hydroxycholesterol on glioma cells.

Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic, deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell death was studied on C6 rat glioma cells, and 7ß-hydroxycholesterol was used as positive cytotoxic control. The highest cytotoxicity was observed with lithocholic and chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5ß-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5ß-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death associated with mitochondrial membrane potential loss and reactive oxygen species overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found with 7ß-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7ß-hydroxycholesterol. Compound 1b and 7ß-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7ß-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1, Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7ß-hydroxycholesterol-induced cell death, respectively, supporting that 7ß-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7ß-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a non apoptotic mode of cell death, involving the mitochondria and associated with several characteristics of autophagy.

Cellular interactions of functionalized superparamagnetic iron oxide nanoparticles on oligodendrocytes without detrimental side effects: Cell death induction, oxidative stress and inflammation.

Iron oxide nanoparticles have the capability to cross Blood Brain Barrier (BBB) and hence are widely investigated for biomedical operations in the central nervous system. Before being used for the biomedical purpose, it is necessary to investigate its biocompatibility, dosimetry and biological interaction. In the present study, in-house synthesized superparamagnetic iron oxide nanoparticles (SPIONs) were functionalized using the polymer, PolyEthylene Glycol (PEG) and a fluorophore (Rhodamine). The interaction of these nanoparticles with murine oligodendrocytes 158N was studied using different assays. The nanoparticles were taken up by the cells via endocytosis and there was a dose-dependent increase in the intracellular iron content as revealed by flow cytometry, transmission electron microscopy and confocal microscopy. Nanoparticles remained stable inside cells even after 24 h. Cell sorting capacity using a magnet depended on the number of particles interact per cell. SPIONs exhibited good biocompatibility as no toxicological responses, including morphological changes, loss of viability, oxidative stress or inflammatory response (IL-1ß, IL-6 secretion) were observed. Together, these data show that the in-house synthesized SPIONs have no side effects on 158N cells, and constitute interesting tools for biomedical applications across brain, including cellular imaging and targeting.

Protective effects of bezafibrate against elaidic acid-induced accumulation of lipid droplets in monocytic cells.

Some factors related to diet, such as trans fatty acids (TFA), are known to be involved in the progression of atherosclerosis in humans. Thus, the aim of our study was (i) to evaluate the effects of three dietary free fatty acids (FFA) (elaidic (EA), oleic (OA) and palmitic acid (PA)) on U937 human monocytes, and (ii) to study the eventual benefits of bezafibrate (BZF), a pan-agonist for PPAR isoforms (α, γ and δ) in U937 cells treated with FFA. Morphologic and functional changes were investigated by microscopic and flow cytometric methods. Cellular lipid content, lipid droplets and FA composition were identified and studied. All analyses were also realized in association with or without BZF. Contrary to OA and PA, EA slightly induced both propidium iodide-positive cells and mitochondrial depolarization. In addition, in contrast to OA and PA, EA induced only a slight increase in superoxide anion production. However, EA and OA promoted cytoplasmic lipid droplets accumulation. Only EA and OA significantly increased CD36 expression. It is noteworthy that BZF had a more or less pronounced protective effect against EA-, OA- and PA-induced side effects: BZF attenuated the impaired cell viability and inflammatory response, decreased superoxide anion production and prevented the accumulation of neutral and polar lipids. The effects were less pronounced with OA and PA than with EA. Altogether, our data revealed a benefit of BZF on the side effects induced especially with EA. It may thus be of interest in preventing the early stages of atherosclerotic plaque formation.

Evaluation of two human dental pulp stem cell cryopreservation methods.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.

Dual effect of docosahexaenoic acid (attenuation or amplification) on C22:0-, C24:0-, and C26:0-induced mitochondrial dysfunctions and oxidative stress on human neuronal SK-N-BE cells.

Increased levels of C22:0, C24:0 and C26:0 were found in cortical lesions of patients with Alzheimer's disease (AD). So, it was of interest to precise the cytotoxic effects of these fatty acids, and to determine whether docosahexaenoic acid (DHA), described to prevent AD, can attenuate their eventual side effects. Human neuronal SK-N-BE cells were cultured in the absence or presence of C22:0, C24:0 or C26:0 (0.1-20 µM) without or with DHA (50-150 µM). C22:0, C24:0 and C26:0 induce an inhibition of cell growth, a loss of Δψm, an overproduction of reactive oxygen species (ROS), a decrease of reduced glutathione, and a lipid peroxidation. DHA attenuates C22:0, C24:0 and C26:0 induced-mitochondrial dysfunctions and/or cell growth inhibition measured with MTT whatever the concentrations considered, whereas it can either decrease or amplify (especially at 150 µM) ROS overproduction. C22:0, C24:0 and C26:0 have neurotoxic activities, and depending on its concentration, DHA attenuates or not fatty acid-induced side effects.

Evaluation of two human dental pulp stem cell cryopreservation methods / Evaluación de dos métodos de criopreservación de células troncales de pulpa dental humana

La pulpa dental es una fuente promisoria de células madre mesenquimales para ser utilizadas en terapia celular y medicina regenerativa. El desarrollo de métodos que permitan almacenar las células madre con mínimo compromiso de la viabilidad celular, capacidad de diferenciación y función es necesario para aplicaciones clínicas e investigación. El objetivo de este estudio fue evaluar si las células troncales depulpa dental humana (hDPSCs) aisladas y criopreservadas durante 1, 7 y 30 días conservan la viabilidad y expresiónde marcadores específicos de células troncales pos crio-preservación. Para esto, las hDPSCs se aislaron de 23pacientes sanos entre 18 y 31 años. La pulpa dental se disoció enzimáticamente, y las células CD105+ se separaron mediante el sistema Miltenyi™. Posteriormente, las hDPSCs se criopreservaron utilizando el método de Kamath y de Papaccio, se evaluó la viabilidad pos crio-preservación porcitometría de flujo (7AAD) y la expresión de marcadores CD105+/ CD73+, CD34-/CD45-. El método de Papaccio, mostró mayor viabilidad celular a los 30 días (59,5 por ciento)comparado con el método de Kamath, a 1 día (65,5%) y 7 días (56%) respectivamente. Se observó mayor expresión de los marcadores CD105+/CD34- a 1 y 7 días pos-criopreservación con el método Kamath y CD105+/CD45- a los 3 tiempos decriopreservación. CD73+ presentó mayor expresión en las hDPSCs a las 24 horas y 7 días con el método de Kamath, y al mes con el método Papaccio. Estos resultados sugieren que las hDPSCs expresan marcadores de células troncales mesenquimales postcriopreservación. Sin embargo el tiempo de criopreservación podría modificar la expresión de los marcadores probablemente por alterarla configuración espacial de las proteínas de membrana celular; o por comprometer a las células a cierto grado de diferenciación.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was toevaluate whether human dental pulp stem cells (hDPSCs)isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system.The hDPSCs were cryopreserved using the Kamath andPapaccio methods. Post-cryopreservation viability wasmeasured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greaterexpression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservationtime may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.

Determination of heavy metal content and lipid profiles in mussel extracts from two sites on the moroccan atlantic coast and evaluation of their biological activities on MIN6 pancreatic cells.

Mussels may concentrate pollutants, with possibly significant side effects on human health. Therefore, mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast (Jorf Lasfar [JL], an industrial site, and Oualidia [OL], a vegetable-growing area), were subjected to biochemical analyses to quantify the presence of heavy metals (Cd, Cr, and Pb) and to establish the lipid profile: fatty acid, cholesterol, oxysterol, phytosterol and phospholipid content. In addition, mussel lipid extracts known to accumulate numerous toxic components were tested on murine pancreatic ß-cells (MIN6), and their biological activities were measured with various flow cytometric and biochemical methods to determine their impacts on cell death induction, organelle dysfunctions (mitochondria, lysosomes, and peroxisomes), oxidative stress and insulin secretion. The characteristics of JL and OL lipid extracts were compared with those of commercially available mussels from Spain (SP) used for human consumption. OL and JL contained heavy metals, high amounts of phospholipids, and high levels of oxysterols; the [(unsaturated fatty acids)/(saturated fatty acids)] ratio, which can be considered a sign of environmental stress leading to lipid peroxidation, was low. On MIN6 cells, JL and OL lipid extracts were able to trigger cell death. This event was associated with overproduction of H2 O2 , increased catalase activity, a decreased GSH level, lipid peroxidation and stimulation of insulin secretion. These effects were not observed with SP lipid extracts. These data suggest that some components from OL and JL lipid extracts might predispose to pancreatic dysfunctions. Epidemiological studies would be needed to assess the global risk on human health and the metabolic disease incidence in a context of regular seafood consumption from the OL and JL areas.

Brain peroxisomes.

Peroxisomes are essential organelles in higher eukaryotes as they play a major role in numerous metabolic pathways and redox homeostasis. Some peroxisomal abnormalities, which are often not compatible with life or normal development, were identified in severe demyelinating and neurodegenerative brain diseases. The metabolic roles of peroxisomes, especially in the brain, are described and human brain peroxisomal disorders resulting from a peroxisome biogenesis or a single peroxisomal enzyme defect are listed. The brain abnormalities encountered in these disorders (demyelination, oxidative stress, inflammation, cell death, neuronal migration, differentiation) are described and their pathogenesis are discussed. Finally, the contribution of peroxisomal dysfunctions to the alterations of brain functions during aging and to the development of Alzheimer's disease is considered.

Evidence of oxidative stress in very long chain fatty acid--treated oligodendrocytes and potentialization of ROS production using RNA interference-directed knockdown of ABCD1 and ACOX1 peroxisomal proteins.

X-linked adrenoleukodystrophy (X-ALD) and pseudo neonatal adrenoleukodystrophy (P-NALD) are neurodegenerative demyelinating diseases resulting from the functional loss of the peroxisomal ATP-binding cassette transporter D (ABCD1) and from single peroxisomal enzyme deficiency (Acyl-CoA oxidase1: ACOX1), respectively. As these proteins are involved in the catabolism of very long chain fatty acids (VLCFA: C24:0, C26:0), X-ALD and P-NALD patients are characterized by the accumulation of VLCFA in plasma and tissues. Since peroxisomes are involved in the metabolism of reactive oxygen species (ROS) and nitrogen species (RNS), we examined the impact of VLCFA on the oxidative status of 158N murine oligodendrocytes expressing or not Abcd1 or Acox1. VLCFA triggers an oxidative stress characterized by an overproduction of ROS and RNS associated with lipid peroxidation, protein carbonylation, increased superoxide dismutase (SOD) activity, decreased catalase activity and glutathione level. SiRNA knockdown of Abcd1 or Acox1 increased ROS and RNS production even in the absence of VLCFA, and especially potentialized VLCFA-induced ROS overproduction. Moreover, mainly in cells with reduced Acox1 level, the levels of VLCFA and neutral lipids were strongly enhanced both in untreated and VLCFA - treated cells. Our data obtained on 158N murine oligodendrocytes highlight that VLCFA induce an oxidative stress, and demonstrate that Abcd1 or Acox1 knockdown contributes to disrupt RedOx equilibrium supporting a link between oxidative stress and the deficiency of Abcd1 or Acox1 peroxisomal proteins.