Abnormal microglial activation in the Cstb^−/− mouse, a model for progressive myoclonus epilepsy, EPM1

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal-recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss-of-function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb(-/-) mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb(-/-) mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb(-/-) mouse brain, expression of the inflammatory marker p-p38 MAPK and the proportion of both pro-inflammatory M1 and anti-inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb(-/-) mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro-inflammatory inducible nitric oxide synthase (iNOS) and anti-inflammatory arginase 1 (ARG1) in Cstb(-/-) mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb(-/-) microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1.

Inhibition of MyD88 Signaling Skews Microglia/Macrophage Polarization and Attenuates Neuronal Apoptosis in the Hippocampus After Status Epilepticus in Mice

“…These findings also support previous reports indicating in different epilepsy models that microglia M1/M2 polarization plays crucial roles in the pathogenesis of epilepsy [23][24][25]. Although the details of M1/M2 polarization differ from model to model, all these studies show that M1 polarization is increased in the early stage after SE even before myoclonus symptoms, which is consistent with the findings in our study.…”

“…Although the details of M1/M2 polarization differ from model to model, all these studies show that M1 polarization is increased in the early stage after SE even before myoclonus symptoms, which is consistent with the findings in our study. M2 polarization has been observed in pilocarpine-induced SE [23,25] and loss-of-function mutations in the cystatin B gene (CSTB) [24] but not in a kainite-induced SE model [23]. M2 microglia were not found to be predominant after pilocarpineinduced SE in our study, which may be due to variations in model establishment, selected time points, and methods used for identifying M1/M2 phenotypes.…”

“…Neuronal excitotoxicity over a large area produces endogenous damage-associated molecular pattern proteins, such as high mobility group box 1 protein (HMGB-1), which activate MG via TLR4 and other innate immune receptors [7,22]. The relationship between MG/ MΦ polarization and seizure induction has attracted some attention but remains unclear as of yet [23][24][25]. If MG/MΦ polarization could be documented, regulating MG/MΦ polarization toward anti-inflammation would be a therapeutic strategy to address epilepsy by protecting the CNS environment.…”

Brain inflammation is accompanied by peripheral inflammation in Cstb −/− mice, a model for progressive myoclonus epilepsy

“…EPM1 is caused by loss-of-function mutations in the cystatin B ( CSTB ) gene [2, 3], which encodes an inhibitor of lysosomal cysteine cathepsins [4]. CSTB is highly expressed in immune cells, e.g., in blood leukocytes, hepatic lymphocytes, placental macrophages, and microglia [5–9], and it is upregulated in vitro by pro-inflammatory stimulation [8, 10, 11]. In immune cells, the function of CSTB has been linked to chemotaxis [8], expression and secretion of cytokines, and release of nitric oxide [10, 12, 13], implying a role in the immune response.…”

“…CSTB is highly expressed in immune cells, e.g., in blood leukocytes, hepatic lymphocytes, placental macrophages, and microglia [5–9], and it is upregulated in vitro by pro-inflammatory stimulation [8, 10, 11]. In immune cells, the function of CSTB has been linked to chemotaxis [8], expression and secretion of cytokines, and release of nitric oxide [10, 12, 13], implying a role in the immune response. CSTB function has also been associated with diverse cellular processes, such as regulation of apoptosis [14, 15], bone resorption [16, 17], protection of neurons from oxidative stress [18], and cell cycle progression [19].…”