Abstract
Microglial calcium signaling is rare in a baseline state but strongly engaged during early epilepsy development. The mechanism(s) governing microglial calcium signaling are not known. By developing an in vivo uridine diphosphate (UDP) fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP can signal through the microglial-enriched P2Y6 receptor to increase calcium activity during epileptogenesis. P2Y6 calcium activity is associated with lysosome biogenesis and enhanced production of NF-kB-related cytokines. In the hippocampus, knockout of the P2Y6 receptor prevents microglia from fully engulfing neurons. Attenuating microglial calcium signaling through calcium extruder (“CalEx”) expression recapitulates multiple features of P2Y6 knockout, including reduced lysosome biogenesis and phagocytic interactions. Ultimately, P2Y6 knockout mice retain more CA3 neurons and better cognitive task performance during epileptogenesis. Our results demonstrate that P2Y6 signaling impacts multiple aspects of myeloid cell immune function during epileptogenesis.
Commentary
Epilepsy affects over 65 million people worldwide. 1 Though there are over 30 medications 2 and a range of other therapies (surgeries, devices, diets, etc), about a third of patients do not achieve adequate seizure control with medications 3 and alternate therapies are only effective in a subset of medication nonresponders. This puts them at great risk for morbidity and mortality, and it contributes immensely to financial and societal cost. 4 While improved therapeutic approaches continue to be investigated, a more definitive approach would be to prevent the development of epilepsy, or epileptogenesis, in the first place. In order to do this, the mechanisms underlying epileptogenesis need to be understood. At present, they are incompletely understood, at best.
Though neurons are the electrically active, excitable cells in the brain, and excitation–inhibition imbalance is the key tenet underlying epilepsy, microglia are increasingly recognized as playing a role in circuit modulation underlying epileptogenesis. 5 One marker of excitation, or generally of cellular activation, is Ca2+ signaling. Since microglia are not typically electrically excitable, at baseline they do not display much appreciable Ca2+ signaling; however, microglia are seemingly capable of increasing Ca2+ signaling in the setting of acute brain insult. 6 In this study, Umpierre et al identify novel mechanisms for aberrant Ca2+ signaling in microglia as a critical mechanism in epileptogenesis. 7 They hypothesized that purinergic signaling mediates this phenomenon. While the focus has been on adenosine triphosphate (ATP), a major microglial signaling molecule, there are many reasons uridine diphosphate (UDP) may play an important role; 8 however, UDP has been understudied due to lack of specific tools to study it. 7
To begin to examine UDP, Umpierre et al first expressed the genetically encoded Ca2+ indicator GCaMP6 in microglia, under the control of the Cx3Cr1 promoter, in brain slices. They demonstrated that UDP increased Ca2+ signaling in microglia in cortex and in CA1 and CA3 hippocampal regions. This effect was blocked with a purine sensitive, P2Y6 receptor antagonist, but not antagonists against P2X4/7 or P2Y12 receptors. In addition, transcripts for P2ry6 were increased in microglia.
Next, to further study a role for UDP, they developed and characterized a novel fluorescent G-protein-coupled receptor activation-based (GRAB) sensor 9 for UDP. In this case the GRAB sensor was a modified chicken P2Y6 receptor conjugated to green fluorescent protein such that it fluoresced when bound to UDP. They then employed this sensor to evaluate UDP release following status epilepticus (SE) in the rat kainic acid post-SE model of epilepsy, describing both distinct temporal and spatial patterns of release. They also evaluated UDP release during the latent period following SE during which epileptogenesis occurs and found that responses were attenuated in P2Y6 receptor knockout mice, suggesting the effect is P2Y6-dependent.
Interestingly, they next demonstrated that P2Y6 activation drives lysosome biogenesis and facilitates phagocytosis, key immunological features associated with microglial activation. Then, they employed a novel calcium extruder to attenuate Ca2+ signaling and found that this mimicked lysosome impairment in microglia. Activation of transcripts in proinflammatory pathways was seen in association with phagocytosis. They also observed increases in inflammatory nuclear factor-kappa B (NF-kB) cytokines interleukin-1beta (IL-1b) and tumor necrosis factor-alpha (TNF-a).
SE and epileptogenesis in the kainic acid model are well known to be associated with cell loss in the CA3 region of the hippocampus. They observed this in WT mice, but found that there was improved survival of CA3 pyramidal cells in P2Y6 knockout mice. Similarly, epilepsy is known to be associated with cognitive impairment. Indeed, WT mice demonstrated cognitive impairment in the novel object recognition task while P2Y6 knockout mice demonstrated preserved cognitive performance.
Here they demonstrate that the UDP-P2Y6 signaling pathway mediates cellular, molecular, behavioral, and cognitive sequelae of SE and that this correlates with timepoints relevant to epileptogenesis in the kainic acid epilepsy model they employed. Severity of SE can vary from animal to animal and can have an effect on development of epilepsy; therefore, to attempt to control for this, mice were only included in the study if they experienced 8 to 12 moderate to severe (Racine grade 4 or 5) seizures during the SE period. As a proxy for SE severity, they examined C-fos expression one day after SE in a cohort of mice and found no difference between genotypes. However, an important next step would be to determine whether there is an effect on the development of epilepsy itself (ie, Do animals still develop spontaneous seizures? Are seizures less severe or less frequent than without P2Y6 inhibition?). It would also be important to know if there are changes in some of the anatomical hallmarks of epileptogenesis in these models, such as hippocampal mossy fiber sprouting, etc. They speculate about potential changes in immune markers that might be involved and discuss the need to explore this further in future studies.
They employed one of several emerging approaches for assessing cellular Ca2+ activity. In this case, a UDP GRAB sensor that they engineered, which allows real-time assessment of activity of a specific molecule (ie, UDP) in the mouse brain. They also employed interesting calcium extruder (CalEx) technology whereby the calcium-extruding protein, ATPase plasma membrane Ca2+ transporting 2 (ATP2B2), was overexpressed to demonstrate that limiting the increase in Ca2+ signaling could limit sequelae of SE.
Here, they examined one model of epileptogenesis which recapitulates many features of the very common temporal lobe epilepsy in patients. Another important next step would be to determine how generalizable their findings might be to other epilepsy models. For instance, epilepsy associated with traumatic brain injury, chronic traumatic encephalopathy, and others.
If the findings are generalizable to epilepsies triggered by a range of insults, their work could inform prophylactic strategies to prevent epilepsy that may occur following certain exposures, injuries, etc. For instance, perhaps there could be a simple pharmacological measure to employ in military personnel before heading off to combat where they could be at high risk for traumatic brain injury associated epilepsy. The caveat to this being that the mechanisms they targeted here are broadly present in the body. Therefore, therapies would likely need to be targeted to specific brain regions to not incur a range of untoward side effects. There are a wide range of tools that could be employed to more specifically study these mechanisms in animal models. And there are several potential means by which this could be accomplished in humans, including the use of nanoparticles.
Nonetheless, Umpierre et al demonstrate the power of developing novel tools to evaluate biologic mechanisms that previously were not amenable to study. In so doing, they uncover an interesting mechanism that is involved in epileptogenesis and represents an exciting, potentially druggable target for the prevention of epilepsy.
