DREADD Microglia? Targeted Microglial Gi Activation in the Acute Phase can Reduce Seizure Severity,but is Detrimental in the Long Term

Abstract

Chemogenetic Approaches Reveal Dual Functions of Microglia in Seizures

Aastha Dheer, Dale B Bosco, Jiaying Zheng, Lingxiao Wang, Shunyi Zhao, Koichiro Haruwaka, Min-Hee Yi, Abhijeet Barath, Dai-Shi Tian, Long-Jun Wu. Brain Behav Immun 2024;115:406–418. PMID: 37926132. doi:10.1016/j.bbi.2023.11.002

Microglia are key players in maintaining brain homeostasis and exhibit phenotypic alterations in response to epileptic stimuli. However, it is still relatively unknown if these alterations are pro- or anti-epileptic. To unravel this dilemma, we employed chemogenetic manipulation of microglia using the artificial Gi-Designer Receptors Exclusively Activated by Designer Drugs (Gi-DREADD) receptor within a kainic acid (KA) induced murine seizure model. Our results indicate that acute Gi-DREADD activation with clozapine N-oxide can reduce seizure severity. Additionally, we observed increased interaction between microglia and neuronal soma, which correlated with reduced neuronal hyperactivity. Interestingly, prolonged activation of microglial Gi-DREADDS by repeated doses of clozapine N-oxide (CNO) over 3 days, arrested microglia in a less active, homeostatic-like state, which is associated with increased neuronal loss after KA-induced seizures. RNAseq analysis revealed that prolonged activation of Gi-DREADD interferes with interferon β signaling and microglia proliferation. Thus, our findings highlight the importance of microglial Gi signaling not only during status epilepticus (SE) but also within later seizure-induced pathology.

Commentary

Microglia, the main immune cells of the brain, play a complex role in both maintaining brain health and contributing to various neurological disorders, including epilepsy. However, the precise roles played by these cells in the generation and resolution of epileptic seizures are still unclear, with studies suggesting both beneficial ¹ as well as detrimental ² effects.

In the study chosen for this Commentary, Dheer et al ³ used chemogenetic (also known as pharmacogenetic) tools to explore the microglial effects in the context of kainic acid-induced seizures. Chemogenetics encompasses cutting-edge versatile tools allowing the mechanistic interrogation of cells within various experimental paradigms and models. This is achieved by cell-specific expression of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that comprise a family of engineered G-protein-coupled receptors (GPCRs) that allow cell-specific stimulation. These receptors could be either human M3 muscarinic receptors (hM3Dq) or hM4Di, activating Gq- or Gi-receptors, respectively. Gq signaling leads to the release of Ca²⁺ from internal storage, thus triggering multiple intracellular signaling processes. ⁴ Gi signaling can lower the intracellular cyclic adenosine monophosphate levels, induce G-protein β/γ-mediated signaling processes, activate G-protein-regulated inward rectifier K⁺ channels, and/or inhibit Ca²⁺ channels. ⁴ Stimulation of DREADDs is selectively achieved by inert exogenous compounds, such as clozapine N-oxide (CNO). ⁵ DREADD-mediated modulation of microglial physiology is a recent development. In microglia, Gq-coupled DREADDs (ie, hM3Dq) increase intracellular calcium, similar to microglial GPCRs, such as purinoceptors P2Y6R, metabotropic glutamate receptors mGluR5 or muscarinic acetylcholine receptors M3R. The effect of Gi-coupled DREADD (ie, hM4Di) activation in microglia is less understood, but it may mimic the effects of microglial Gi-coupled GPCRs, such as α2-adrenergic receptors, P2Y12R, P2Y13R or GABA_B receptors (for review, see Bossuyt et al ⁴ ).

In their study, Dheer et al ³ used a cross of mice (Cx3cr1^CreER/WT: R26^{LSL−hM4Di/WT}) expressing hM4Di under the control of the immune Cx3cr1 promoter. Thus, the expression of hM4Di in the brain was restricted to microglial cells, although, as noted, it is also expected to be expressed in peripheral immune cells, potentially mediating some of the effects recorded in the study. These mice were compared with control mice not expressing hM4Di (Cx3cr1^CreER/WT). To induce seizures, the mice were administered kainate intracerebroventricularly, and to modulate microglial functions through hM4Di, they then received CNO i.p. To dissect the time-dependent role of microglial cells, the brains of these mice were analyzed at specific time points: 2 h postkainate administration (achieving acute modulation of microglial physiology) and 3 days postkainate administration following daily CNO modulation of microglia physiology. Acute hM4Di signaling in microglia lessened the severity of seizures, whereas prolonged hM4Di signaling resulted in the immune cells becoming trapped in a chronic homeostatic-like state, which coincided with increased neuronal loss and degeneration in the CA3 hippocampal brain region typically affected by seizures. Thus, one of the key findings of the study highlights the paradoxical double-edged sword of microglial roles in epilepsy.

Dheer et al ³ observed that acute modulation of microglial physiology through hM4Di activation attenuated seizure severity and increased microglia–neuron soma interaction within the hippocampus. Their results also revealed an increase in CD68⁺ phagocytic Iba1⁺ cells within 2 h from the initiation of the kainate insult, raising questions if all the Iba1⁺ cells quantified in the study were of microglial origin or if this increase reflects infiltrated peripheral immune cells. Chronic hM4Di stimulation of microglia, however, led to a decrease in levels of both Iba1⁺ and Iba1⁺ CD68⁺ cells, together with a decrease in the proliferating CD11b⁺ cells (also a marker of both microglia and peripheral immune cells). To address the problem of infiltrating immune cells, Dheer et al ³ used CD169 (a marker of peripheral monocytes) and TMEM119 (a more specific marker of microglial cells); however, these results remain inconclusive. In another study using chronic inhibition of microglial hM4Di signaling through microglia-specific expression of pertussis toxin, investigators observed decreased microglia-neuron interactions, increased hypersynchrony, and spontaneous seizures after evoked physiological activity. ⁶ Although divergent in their experimental paradigms, these studies reveal the detrimental effects of “freezing” microglial cells in a particular physiological state.

An important point raised by the study from the Wu laboratory ³ is the time window in which the physiology of microglia would have to be modulated for translational purposes. Although in the initial phase, microglial reactivity during and directly after seizure had damaging effects and required inhibition, 3 days of prolonged stimulation of microglial hM4Di contributed to neurodegeneration and neuronal loss. As Gi mediates signaling of various receptors, including purinergic, glutamatergic, and adenosine-sensing receptors in microglial cells,^6,7 hM4Di stimulation of microglia could have far-reaching physiological effects on the brain. In a previous study, ⁶ G-protein-coupled Rho GTPases, which regulate cytoskeletal rearrangement and motility downstream of Gi activation, were found to be important in the modulation of microglia–neuron interactions and network hypersynchrony. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular adenosine triphosphate (ATP), which is released upon neuronal activation by neurons and astrocytes. ⁸ Within the context of epilepsy, an earlier study from the Wu laboratory ⁹ revealed that the absence of P2Y12R, a microglial-specific Gi-coupled receptor that senses ATP release, reduced seizure-induced increases in microglial process numbers and worsened kainate-induced seizures. Dissecting which of these Gi-coupled receptors and which second messenger signaling pathways are involved could have translational value (not only for epilepsy), especially as no true microglia-specific treatment has emerged so far. In addition, capturing the full spectrum of microglial function during chronic epilepsy, a more complex scenario, is still challenging to achieve, as also acknowledged by the present study. Utilizing models that better mimic chronic epilepsy could bring further valuable insights into the role of microglia in spontaneous recurrent seizures and epileptogenesis itself.

Dheer et al ³ study also highlights the need for further exploration of the precise microglial signaling pathways active in the epileptic brain. Using RNA sequencing, the study identified molecules such as interferon-beta (IFN-β) and interleukin-1 alpha (IL-1α) as important microglial players following chronic hM4Di stimulation of microglia. If these changes in messenger RNA levels lead to differences in protein expression (as partially shown in the study), this points to a potential additional way of microglial response to insults induced by seizures, diverging from the classic IL-1β/tumor necrosis factor-α (TNF-α)-mediated responses observed in other epilepsy studies.^7,10

The biphasic paradoxical microglial responses are now well-established in other neurological conditions, including epilepsy. ² Nevertheless, the study of Dheer et al ³ emphasizes the importance of considering the timing and context of microglial activity when designing future epilepsy treatment interventions. It also acknowledges that other cell types, such as astrocytes, contribute to neuroinflammation and neuronal death in epilepsy. ¹⁰ While this study focused on microglia, a complete understanding of epilepsy will likely require investigating the interplay between various cell types in the brain.