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.

Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.

Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy.

Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing α-synuclein oligomeric species toxicity.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

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.