Esteban Oyarzabal

Microglial regulation of immunological and neuroprotective functions of astroglia

Microglia and astroglia play critical roles in the development, function, and survival of neurons in the CNS. However, under inflammatory conditions the role of astrogliosis in the inflammatory process and its effects on neurons remains unclear. Here, we used several types of cell cultures treated with the bacterial inflammogen LPS to address these questions. We found that the presence of astroglia reduced inflammation‐driven neurotoxicity, suggesting that astrogliosis is principally neuroprotective. Neutralization of supernatant glial cell line‐derived neurotrophic factor (GDNF) released from astroglia significantly reduced this neuroprotective effect during inflammation. To determine the immunological role of astroglia, we optimized a highly‐enriched astroglial culture protocol and demonstrated that LPS failed to induce the synthesis and release of TNF‐α and iNOS/NO. Instead we found significant enhancement of TNF‐α and iNOS expression in highly‐enriched astroglial cultures required the presence of 0.5–1% microglia, respectively. Thus suggesting that microglial‐astroglial interactions are required for LPS to induce the expression of pro‐inflammatory factors and GDNF from astroglia. Specifically, we found that microglia‐derived TNF‐α plays a pivotal role as a paracrine signal to regulate the neuroprotective functions of astrogliosis. Taken together, these findings suggest that astroglia may not possess the ability to directly recognize the innate immune stimuli LPS, but rather depend on crosstalk with microglia to elicit release of neurotrophic factors as a counterbalance to support neuronal survival from the collateral damage generated by activated microglia during neuroinflammation. GLIA 2015;63:118–131

Post-treatment with an ultra-low dose of NADPH oxidase inhibitor diphenyleneiodonium attenuates disease progression in multiple Parkinson's disease models.

Nicotinamide adenine dinucleotide phosphate oxidase, a key superoxide-producing enzyme, plays a critical role in microglia-mediated chronic neuroinflammation and subsequent progressive dopaminergic neurodegeneration in Parkinsons disease. Although nicotinamide adenine dinucleotide phosphate oxidase-targeting anti-inflammatory therapy for Parkinsons disease has been proposed, its application in translational research remains limited. The aim of this study was to obtain preclinical evidence supporting this therapeutic strategy by testing the efficacy of an ultra-low dose of the nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium in both endotoxin (lipopolysaccharide)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using post-treatment regimens. Our data revealed that post-treatment with diphenyleneiodonium significantly attenuated progressive dopaminergic degeneration and improved rotarod activity. Remarkably, post-treatment with diphenyleneiodonium 10 months after lipopolysaccharide injection when mice had 30% loss of nigral dopaminergic neurons, showed high efficacy in protecting the remaining neuronal population and restoring motor function. Diphenyleneiodonium-elicited neuroprotection was associated with the inhibition of microglial activation, a reduction in the expression of proinflammatory factors and an attenuation of α-synuclein aggregation. A pathophysiological evaluation of diphenyleneiodonium-treated mice, including assessment of body weight, organs health, and neuronal counts, revealed no overt signs of toxicity. In summary, infusion of ultra-low dose diphenyleneiodonium potently reduced microglia-mediated chronic neuroinflammation by selectively inhibiting nicotinamide adenine dinucleotide phosphate oxidase and halted the progression of neurodegeneration in mouse models of Parkinsons disease. The robust neuroprotective effects and lack of apparent toxic side effects suggest that diphenyleneiodonium at ultra-low dose may be a promising candidate for future clinical trials in Parkinsons disease patients.

Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinsons disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1−/−), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose–response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91phox and inducing membrane translocation of the cytosolic subunits p47phox and p67phox. The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD.

Critical role of the Mac1/NOX2 pathway in mediating reactive microgliosis-generated chronic neuroinflammation and progressive neurodegeneration.

As average life expectancy rises throughout the world, neurodegenerative diseases have emerged as one of the greatest global public heath challenges in modern times. Substantial efforts have been made in researching neurodegenerative diseases over the last few decades, yet their predominantly sporadic nature has made uncovering their etiologies challenging. Mounting evidence has suggested that factors like damage-associated molecular patterns (DAMPs) released by stressed and dying neurons are likely involved in disease pathology and in stimulating chronic activation of microglia that contributes to neuronal oxidative stress and degeneration. This review focuses on how the microglial integrin receptor Mac1 and its downstream effector NADPH oxidase (NOX2) contribute to maintaining chronic neuroinflammation and are crucial in inflammation-driven neurotoxicity in neurodegenerative diseases. Our hope is to provide new insights on novel targets and therapies that could slow or even halt neurodegeneration.

Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases

Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinsons disease (PD). The inhibition of NOX2‐generated superoxide has become an effective strategy for developing disease‐modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long‐acting NOX2 inhibitor. However, due to its non‐specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra‐low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra‐low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10−14 and 10−13 M) displays no toxicity in primary midbrain neuron‐glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)‐induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein‐containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post‐treatment study. When subpicomolar DPI was added to neuron‐glia cultures pretreated with lipopolysaccharide, 1‐methyl‐4‐phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPIs unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post‐treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials. GLIA 2014;62:2034–2043

Preparation of Rodent Primary Cultures for Neuron–Glia, Mixed Glia, Enriched Microglia, and Reconstituted Cultures with Microglia

Microglia, neurons, and macroglia (astrocytes and oligodendrocytes) are the major cell types in the central nervous system. In the past decades, primary microglia-enriched cultures have been widely used to study the biological functions of microglia in vitro. In order to study the interactions between microglia and other brain cells, neuron-glia, neuron-microglia, and mixed glia cultures were developed. The aim of this chapter is to provide basic and adaptable protocols for the preparation of these microglia-containing primary cultures from rodent. Meanwhile, we also want to provide a collection of tips from our collective experiences doing primary brain cell cultures.

A novel role of microglial NADPH oxidase in mediating extra‐synaptic function of norepinephrine in regulating brain immune homeostasis

Although the peripheral anti‐inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti‐inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti‐inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation‐based dopaminergic neurotoxicity models. In mice, NE‐depleting toxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) was injected at 6 months of lipopolysaccharide (LPS)‐induced neuroinflammation. It was found that NE depletion enhanced LPS‐induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose‐dependently protected dopaminergic neurons from LPS‐induced neurotoxicity by inhibiting microglia activation and subsequent release of pro‐inflammatory factors. However, NE‐elicited neuroprotection was not totally abolished in cultures from β2‐adrenergic receptor (β2‐AR)‐deficient mice, suggesting that novel pathways other than β2‐AR are involved. To this end, It was found that submicromolar NE dose‐dependently inhibited NADPH oxidase (NOX2)‐generated superoxide, which contributes to the anti‐inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (−) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS‐induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism. GLIA 2015;63:1057–1072

Loss of Brain Norepinephrine Elicits Neuroinflammation-Mediated Oxidative Injury and Selective Caudo-Rostral Neurodegeneration

Environmental toxicant exposure has been strongly implicated in the pathogenesis of Parkinson’s disease (PD). Clinical manifestations of non-motor and motor symptoms in PD stem from decades of progressive neurodegeneration selectively afflicting discrete neuronal populations along a caudo-rostral axis. However, recapitulating this spatiotemporal neurodegenerative pattern in rodents has been unsuccessful. The purpose of this study was to generate such animal PD models and delineate mechanism underlying the ascending neurodegeneration. Neuroinflammation, oxidative stress, and neuronal death in mice brains were measured at different times following a single systemic injection of lipopolysaccharide (LPS). We demonstrate that LPS produced an ascending neurodegeneration that temporally afflicted neurons initially in the locus coeruleus (LC), followed by substantia nigra, and lastly the primary motor cortex and hippocampus. To test the hypothesis that LPS-elicited early loss of noradrenergic LC neurons may underlie this ascending pattern, we used a neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to deplete brain norepinephrine. DSP-4 injection resulted in a time-dependent ascending degenerative pattern similar to that generated by the LPS model. Mechanistic studies revealed that increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX2)-dependent superoxide/reactive oxygen species (ROS) production plays a key role in both LPS- and DSP-4-elicited neurotoxicity. We found that toxin-elicited chronic neuroinflammation, oxidative neuronal injuries, and neurodegeneration were greatly suppressed in mice deficient in NOX2 gene or treated with NOX2-specific inhibitor. Our studies document the first rodent PD model recapturing the ascending neurodegenerative pattern of PD patients and provide convincing evidence that the loss of brain norepinephrine is critical in initiating and maintaining chronic neuroinflammation and the discrete neurodegeneration in PD.

Neuroinflammation in Neurological Dysfunction and Degeneration

The inflammatory hypothesis states that neuroinflammation originating from infection, trauma, or toxicant exposures may alter brain immunohomeostasis, culminating in neurocircuitry dysfunction and progressive degeneration. Neuroinflammation can accelerate the progression of neurodegenerative diseases, yet many argue that although it is a necessary element, it is not a sufficient explanation for all of the features associated with neurodegenerative diseases—citing atypical disease-specific protein lesions, the failures of anti-inflammatory treatments, and prevalence mismatch for neurodegenerative diseases linked to infection or chronic inflammatory disorders. Thus, whether neuroinflammation in neurodegenerative diseases is pathogenic or exists to mitigate neuronal dysfunction and degeneration is still being debated. This chapter will evaluate the inflammatory hypothesis and provide insight into how environmentally derived neuroinflammation may alter neurological function and promote neuronal loss in neurodegenerative diseases.