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.

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.

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.

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.

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.