Parkinson's disease-derived α-synuclein assemblies combined with chronic-type inflammatory cues promote a neurotoxic microglial phenotype.

Parkinson's disease (PD) is a common age-related neurodegenerative disorder characterized by the aggregation of α-Synuclein (αSYN) building up intraneuronal inclusions termed Lewy pathology. Mounting evidence suggests that neuron-released αSYN aggregates could be central to microglial activation, which in turn mounts and orchestrates neuroinflammatory processes potentially harmful to neurons. Therefore, understanding the mechanisms that drive microglial cell activation, polarization and function in PD might have important therapeutic implications. Here, using primary microglia, we investigated the inflammatory potential of pure αSYN fibrils derived from PD patients. We further explored and characterized microglial cell responses to a chronic-type inflammatory stimulation combining PD patient-derived αSYN fibrils (FPD), Tumor necrosis factor-α (TNFα) and prostaglandin E2 (PGE2) (TPFPD). We showed that FPD hold stronger inflammatory potency than pure αSYN fibrils generated de novo. When combined with TNFα and PGE2, FPD polarizes microglia toward a particular functional phenotype departing from FPD-treated cells and featuring lower inflammatory cytokine and higher glutamate release. Whereas metabolomic studies showed that TPFPD-exposed microglia were closely related to classically activated M1 proinflammatory cells, notably with similar tricarboxylic acid cycle disruption, transcriptomic analysis revealed that TPFPD-activated microglia assume a unique molecular signature highlighting upregulation of genes involved in glutathione and iron metabolisms. In particular, TPFPD-specific upregulation of Slc7a11 (which encodes the cystine-glutamate antiporter xCT) was consistent with the increased glutamate response and cytotoxic activity of these cells toward midbrain dopaminergic neurons in vitro. Together, these data further extend the structure-pathological relationship of αSYN fibrillar polymorphs to their innate immune properties and demonstrate that PD-derived αSYN fibrils, TNFα and PGE2 act in concert to drive microglial cell activation toward a specific and highly neurotoxic chronic-type inflammatory phenotype characterized by robust glutamate release and iron retention.

Microglial glutamate release evoked by α-synuclein aggregates is prevented by dopamine.

When activated, microglial cells have the potential not only to secrete typical proinflammatory mediators but also to release the neurotransmitter glutamate in amounts that may promote excitotoxicity. Here, we wished to determine the potential of the Parkinson's disease (PD) protein α-Synuclein (αS) to stimulate glutamate release using cultures of purified microglial cells. We established that glutamate release was robustly increased when microglial cultures were treated with fibrillary aggregates of αS but not with the native monomeric protein. Promotion of microglial glutamate release by αS aggregates (αSa) required concomitant engagement of TLR2 and P2X7 receptors. Downstream to cell surface receptors, the release process was mediated by activation of a signaling cascade sequentially involving phosphoinositide 3-kinase (PI3K) and NADPH oxidase, a superoxide-producing enzyme. Inhibition of the Xc- antiporter, a plasma membrane exchange system that imports extracellular l-cystine and exports intracellular glutamate, prevented the release of glutamate induced by αSa, indicating that system Xc- was the final effector element in the release process downstream to NADPH oxidase activation. Of interest, the stimulation of glutamate release by αSa was abrogated by dopamine through an antioxidant effect requiring D1 dopamine receptor activation and PI3K inhibition. Altogether, present data suggest that the activation of microglial cells by αSa may possibly result in a toxic build-up of extracellular glutamate contributing to excitotoxic stress in PD. The deficit in dopamine that characterizes this disorder may further aggravate this process in a vicious circle mechanism.

Parkin deficiency modulates NLRP3 inflammasome activation by attenuating an A20-dependent negative feedback loop.

Neuroinflammation and mitochondrial dysfunction, key mechanisms in the pathogenesis of Parkinson's disease (PD), are usually explored independently. Loss-of-function mutations of PARK2 and PARK6, encoding the E3 ubiquitin protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, account for a large proportion of cases of autosomal recessive early-onset PD. PINK1 and Parkin regulate mitochondrial quality control and have been linked to the modulation of innate immunity pathways. We report here an exacerbation of NLRP3 inflammasome activation by specific inducers in microglia and bone marrow-derived macrophages from Park2-/- and Pink1-/- mice. The caspase 1-dependent release of IL-1ß and IL-18 was, therefore, enhanced in Park2-/- and Pink1-/- cells. This defect was confirmed in blood-derived macrophages from patients with PARK2 mutations and was reversed by MCC950, which specifically inhibits NLRP3 inflammasome complex formation. Enhanced NLRP3 signaling in Parkin-deficient cells was accompanied by a lack of induction of A20, a well-known negative regulator of the NF-κB pathway recently shown to attenuate NLRP3 inflammasome activity. We also found an inverse correlation between A20 abundance and IL-1ß release, in human macrophages challenged with NLRP3 inflammasome inducers. Overall, our observations suggest that the A20/NLRP3-inflammasome axis participates in the pathogenesis of PARK2-linked PD, paving the way for the exploration of its potential as a biomarker and treatment target.

Cannabidiol prevents haloperidol-induced vacuos chewing movements and inflammatory changes in mice via PPARγ receptors.

The chronic use of drugs that reduce the dopaminergic neurotransmission can cause a hyperkinetic movement disorder called tardive dyskinesia (TD). The pathophysiology of this disorder is not entirely understood but could involve oxidative and neuroinflammatory mechanisms. Cannabidiol (CBD), the major non-psychotomimetic compound present in Cannabis sativa plant, could be a possible therapeutic alternative for TD. This phytocannabinoid shows antioxidant, anti-inflammatory and antipsychotic properties and decreases the acute motor effects of classical antipsychotics. The present study investigated if CBD would attenuate orofacial dyskinesia, oxidative stress and inflammatory changes induced by chronic administration of haloperidol in mice. Furthermore, we verified in vivo and in vitro (in primary microglial culture) whether these effects would be mediated by PPARγ receptors. The results showed that the male Swiss mice treated daily for 21 days with haloperidol develop orofacial dyskinesia. Daily CBD administration before each haloperidol injection prevented this effect. Mice treated with haloperidol showed an increase in microglial activation and inflammatory mediators in the striatum. These changes were also reduced by CBD. On the other hand, the levels of the anti-inflammatory cytokine IL-10 increased in the striatum of animals that received CBD and haloperidol. Regarding oxidative stress, haloperidol induced lipid peroxidation and reduced catalase activity. This latter effect was attenuated by CBD. The combination of CBD and haloperidol also increased PGC-1α mRNA expression, a co-activator of PPARγ receptors. Pretreatment with the PPARγ antagonist, GW9662, blocked the behavioural effect of CBD in our TD model. CBD also prevented LPS-stimulated microglial activation, an effect that was also antagonized by GW9662. In conclusion, our results suggest that CBD could prevent haloperidol-induced orofacial dyskinesia by activating PPARγ receptors and attenuating neuroinflammatory changes in the striatum.

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1ß and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology.

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau(P301L), and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau(P301L), that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.

Self-evolving oxidative stress with identifiable pre- and postmitochondrial phases in PC12 cells.

During the neurodegenerative process in several brain diseases, oxidative stress is known to play important roles in disease severity and evolution. Although early events of stress, such as increased lipid peroxidation and decreased superoxide dismutase, are known to characterize early onsets of these diseases, little is known about the events that participate in maintaining the chronic evolving phase influencing the disease progression in neurons. Here, we used differentiated PC12 cells to identify premitochondrial and postmitochondrial events occurring during the oxidative stress cascade leading to apoptosis. Our data indicate that an acute and strong oxidative impulse (500 µM H(2)O(2), 30 min) can induce, in this model, a 24-hr self-evolving stress, which advances from a premitochondrial phase characterized by lysosomes and cathepsin B and D translocations to cytosol and early mitochondrial membrane hyperpolarization. This phase lasts for about 5 hr and is followed by a postmitochondrial phase distinguished by mitochondrial membrane depolarization, reactive oxygen species increase, caspase-9 and caspase-3 activations, and apoptosis. Inhibition of cathepsins B and D suggests that cells can be protected at the premitochondrial phase of stress evolution and that new cathepsins regulators, such as glycosaminoglycans mimetics, can be considered as new therapeutic prototypes for neurodegeneration. Insofar as early oxidative stress markers have been related to the early onset of neurodegeneration, strategies protecting cells at the premitochondrial phase of oxidative stress may have important therapeutic applications.

A miniaturized genotoxicity evaluation system for fast biomaterial-related risk assessment.

Implants and prostheses are widely used to either repair damaged tissues or treat different diseases. Before an implant reaches the market, multiple preclinical and clinical tests must be performed. Along with cytotoxicity or hemocompatibility preclinical tests, genotoxicity is an essential feature to investigate. Indeed, the materials used for implantation should be non-genotoxic, i.e. they should not promote mutations that can potentially lead to tumour formation. However, given the complexity level of genotoxicity tests, such tests are not readily available to biomaterials researchers, which is the reason why this aspect is severely underreported in the literature. To solve this problem, we developed a simplified genotoxicity test that can be further adapted by standard biomaterials laboratories. We started by simplifying the classic Ames test in Petri dishes, after which we developed a miniaturized test in a microfluidic chip, which takes only 24 hours, requiring significantly less material and space. An automatization option with a customized testing chamber architecture and microfluidics-based control system has been designed as well. This optimized microfluidic chip system can significantly improve the availability of genotoxicity tests for biomaterials developers, with the additional benefit of more in-depth observation and quantitative comparison due to the availability of processable image components.

Droplet-based microfluidics platform for antifungal analysis against filamentous fungi.

Fungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. The conventional antifungal screening techniques, such as water agar and 96-well plates, are based on laborious protocols and bulk analysis, restricting the analysis at the single spore level and are time consuming. In this study, we present a droplet-based microfluidic platform that enables antifungal analysis of single spores of filamentous fungus Alternaria alternata. A droplet-based viability assay was developed, allowing the germination and hyphal growth of single A. alternata spores within droplets. The viability was demonstrated over a period of 24 h and the antifungal screening was achieved using Kunshi/Tezuma as antifungal agent. The efficacy results of the droplet-based antifungal analysis were compared and validated with the results obtained from conventional protocols. The percentage inhibitions assessed by the droplet-based platform were equivalent with those obtained by the other two methods, and the Pearson correlation analysis showed high correlation between the three assays. Taken together, this droplet-based microfluidic platform provides a wide range of potential applications for the analysis of fungicide resistance development as well as combinatorial screening of other antimicrobial agents and even antagonistic fungi.

Exploiting the therapeutic potential of ready-to-use drugs: Repurposing antibiotics against amyloid aggregation in neurodegenerative diseases.

Neurodegenerative diseases are chronic and progressive disorders that affect specific regions of the brain, causing gradual disability and suffering that results in a complete inability of patients to perform daily functions. Amyloid aggregation of specific proteins is the most common biological event that is responsible for neuronal death and neurodegeneration in various neurodegenerative diseases. Therapeutic agents capable of interfering with the abnormal aggregation are required, but traditional drug discovery has fallen short. The exploration of new uses for approved drugs provides a useful alternative to fill the gap between the increasing incidence of neurodegenerative diseases and the long-term assessment of classical drug discovery technologies. Drug re-profiling is currently the quickest possible transition from bench to bedside. In this way, experimental evidence shows that some antibiotic compounds exert neuroprotective action through anti-aggregating activity on disease-associated proteins. The finding that many antibiotics can cross the blood-brain barrier and have been used for several decades without serious toxic effects makes them excellent candidates for therapeutic switching towards neurological disorders. The present review is, to our knowledge, the first extensive evaluation and analysis of the anti-amyloidogenic effect of different antibiotics on well-known disease-associated proteins. In addition, we propose a common structural signature derived from the antiaggregant antibiotic molecules that could be relevant to rational drug discovery.

Identification of a Novel 1,4,8-Triazaphenanthrene Derivative as a Neuroprotectant for Dopamine Neurons Vulnerable in Parkinson's Disease.

Parkinson's disease (PD) is a chronic degenerative disorder characterized by typical motor symptoms caused by the death of dopamine (DA) neurons in the midbrain and ensuing shortage of DA in the striatum, at the level of nerve terminals. No curative treatment is presently available for PD in clinical practice. In our search for neuroprotectants in PD, we generated new 1,4,8-triazaphenanthrenes by combining 6-endo-dig-cycloisomerization of propargylquinoxalines and Suzuki or Sonogashira cross-coupling reactions. Neuroprotection assessment of newly synthesized 1,4,8-triazaphenanthrenes in a PD cellular model resulted in the discovery of a new hit compound PPQ (5m). Neuroprotection by 5m was concentration-dependent and the result of a combined effect on intracellular calcium release channels and astroglial cells. Of interest, 5m also counteracted DA cell loss in a mouse model of PD, making this molecule a promising candidate for PD treatment.

Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species.

Synucleinophaties are progressive neurodegenerative disorders with no cure to date. An attractive strategy to tackle this problem is repurposing already tested safe drugs against novel targets. In this way, doxycycline prevents neurodegeneration in Parkinson models by modulating neuroinflammation. However, anti-inflammatory therapy per se is insufficient to account for neuroprotection. Herein we characterise novel targets of doxycycline describing the structural background supporting its effectiveness as a neuroprotector at subantibiotic doses. Our results show that doxycycline reshapes α-synuclein oligomers into off-pathway, high-molecular-weight species that do not evolve into fibrils. Off-pathway species present less hydrophobic surface than on-pathway oligomers and display different ß-sheet structural arrangement. These structural changes affect the α-synuclein ability to destabilize biological membranes, cell viability, and formation of additional toxic species. Altogether, these mechanisms could act synergically giving novel targets for repurposing this drug.

New 6-Aminoquinoxaline Derivatives with Neuroprotective Effect on Dopaminergic Neurons in Cellular and Animal Parkinson Disease Models.

Parkinson's disease (PD) is a neurodegenerative disorder of aging characterized by motor symptoms that result from the loss of midbrain dopamine neurons and the disruption of dopamine-mediated neurotransmission. There is currently no curative treatment for this disorder. To discover druggable neuroprotective compounds for dopamine neurons, we have designed and synthesized a second-generation of quinoxaline-derived molecules based on structure-activity relationship studies, which led previously to the discovery of our first neuroprotective brain penetrant hit compound MPAQ (5c). Neuroprotection assessment in PD cellular models of our newly synthesized quinoxaline-derived compounds has led to the selection of a better hit compound, PAQ (4c). Extensive in vitro characterization of 4c showed that its neuroprotective action is partially attributable to the activation of reticulum endoplasmic ryanodine receptor channels. Most interestingly, 4c was able to attenuate neurodegeneration in a mouse model of PD, making this compound an interesting drug candidate for the treatment of this disorder.

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways.

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson's disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1ß). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

Glycosaminoglycans, protein aggregation and neurodegeneration.

A number of neurodegenerative diseases, as Parkinson, prion, and Alzheimer's diseases, has been directly associated with altered conformations of certain peptides or proteins that assemble to form highly organized aggregates, also called amyloid fibers. Glycosaminoglycans have shown to play important roles on fibrils formation, stability and resistance to proteolysis. This manuscript reviews from basic concepts on the biochemistry and biology of glycosaminoglycans to their implications in neurodegeneration with particular emphasis in pathologic protein aggregation. Prion protein, Aß42, Tau, and α-synuclein, are all proteins that can interact with glycosaminoglycans. We document here how these interactions may modify protein conformation, aggregation kinetics, and fibers stabilization with important consequences in disease. We also raise questions which answers may make advance the understanding of the implication of GAGs in neurodegeneration.