Research PaperThe anti-parkinsonian drug zonisamide reduces neuroinflammation: Role of microglial Nav 1.6
Introduction
Neuroinflammation and microglial activation contributes to the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD) (Bronzuoli et al., 2016; Heneka et al., 2015; Tansey and Goldberg, 2010; Wang et al., 2015). Microglia play an active role in neuroprotection and repair of neurons in the central nervous system following toxic insult and neuronal injury. However, persistent activation of microglia can mediate neuronal death and neurodegeneration by increasing the secretion of inflammatory molecules and cytokines, including tumor necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) (Harrigan et al., 2008; Liu et al., 2010). Given the association between microglial activation and neurodegeneration, a significant amount of effort has been focused on identifying interventions that can dampen the neuroinflammatory response and slow the initiation or progression of neurodegeneration. Unfortunately, these interventions have not yet been successful in clinical trials.
Microglia express a number of ion channels, including Na+ channels that regulate various aspects of inflammatory process, providing a potential target for intervention (Black et al., 2009; Hossain et al., 2017; Pappalardo et al., 2016; Richardson and Hossain, 2013). Although generation of action potentials is the primary function of voltage-gated sodium channels (VGSC), several recent studies demonstrated that VGSC can regulate a number of cellular functions such as morphological transformation, migration, and phagocytosis of microglia when stimulated with lipopolysaccharide (LPS) (Black et al., 2009; Stevens et al., 2013), suggesting potential immunomodulatory properties of VGSC. We recently reported that Na+ influx through VGSC initiates activation of microglia and subsequently triggers an inflammatory pathway by accumulation of intracellular sodium [(Na+)i] after exposure to LPS (Hossain et al., 2013) and pyrethroid insecticides (Hossain et al., 2017), which appears to involve Nav 1.6. The Nav 1.6 isoform is one of most abundantly expressed isoforms within peripheral and central nervous system of the adult (Goldin, 2001; Krzemien et al., 2000; Tzoumaka et al., 2000) and is also found to be a predominant isoform in microglia (Black et al., 2009; Black and Waxman, 2012; Hossain et al., 2017; Hossain et al., 2013). Additional studies from Waxman's group demonstrated that mice exhibit a significant upregulation of Nav 1.6 in activated microglia in an experimental inflammatory/demyelinating model of multiple sclerosis (Craner et al., 2005). Furthermore, they reported that the expression of Nav 1.6 expression increases with morphological transformation of microglia to an amoeboid like appearance. Together, these data suggest that microglial Nav 1.6 plays an important role in regulation of microglial inflammation and could serve as a potential therapeutic target in neurodegeneration.
Zonisamide is an anti-convulsant drug approved by the FDA for the treatment of epilepsy (Sonsalla et al., 2010). The primary mechanism by which zonisamide is thought to exert its anti-epileptic effect is through inhibition of the voltage-gated sodium and T-type calcium channels (Biton, 2007; Kito et al., 1996; Matar et al., 2009; Okada et al., 2002). Recently, zonisamide has been reported to improve symptoms in PD patients when used in combination with other anti-parkinsonian drugs, likely through its ability to inhibit monoamine oxidase B (MAO-B) (Murata et al., 2007). In preclinical models, zonisamide has been shown to provide neuroprotection against seizure (Mares, 2010; Ueda et al., 2005) and ischemia (Minato et al., 1997; Owen et al., 1997). Pre-treatment of mice with zonisamide attenuated the MPTP-induced reduction in striatal DA, DOPAC, and tyrosine hydroxylase (TH), which was likely the result of MAO-B inhibition and decreased MPP+ formation (Sonsalla et al., 2010). More recent studies demonstrate that zonisamide can be protective when given after MPTP administration (Choudhury et al., 2011). However, the mechanisms responsible for this effect have not been fully elucidated.
In the present study, we investigated the expression of microglial Nav1.6 in PD brain and MPTP mice. We further assessed the ability of zonisamide to prevent neuroinflammation in an acute mouse model of neuroinflammation produced by a single injection of MPTP (12.5 mg/kg, s.c.) that results in striatal injury and neuroinflammation within 12 h after MPTP administration (O'Callaghan et al., 1990). Our findings demonstrate that Nav1.6 expression is increased in microglia in PD brain and in mice treated with MPTP. Repeated zonisamide treatment administered post-MPTP significantly reduced this expression, which was accompanied by reduction of microglial activation and associated pro-inflammatory mediators. Thus, in addition to its effects on MAO-B, zonisamide may also have a neuroprotective role in PD by reducing microglial activation through down-regulation of Nav1.6.
Section snippets
Case selection and neuropathological assessment
Specimens were obtained from the Emory Alzheimer Disease Research Center Neuropathology Core, Atlanta, GA. Tissues were fixed in 4% paraformaldehyde for 1 to 2 weeks before being transferred to cryopreservative. The neuropathologic diagnosis of PD was based on the presence of nigral degeneration and Lewy bodies. Control cases had no clinical history or neuropathologic diagnosis of neurologic disease. Case descriptions are provided in Supplemental Table 1.
Immunofluorescent staining for human tissues
Fixed, cryopreserved, free-floating,
Increased expression of Nav 1.6 in striatal microglia in Parkinson's disease patients
Because increased markers of inflammation and microglial activation have been observed in the post mortem brains of PD patients (McGeer et al., 1988) and Nav 1.6 has been found to be a significant contributor to microglial activation in vitro (Black et al., 2009; Hossain et al., 2017; Hossain et al., 2013), we examined Nav 1.6 and microglial activation in the striatum of post mortem brains from PD patients by immunofluorescence staining (Fig. 1; Fig. S1). To this end, we used Iba1 to
Discussion
Chronic neuroinflammation is a common feature of many neurodegenerative diseases and activation of microglia have been proposed to play both causative and aggravating roles in the pathogenic process of DA neuron degeneration in PD (Hirsch et al., 2012; Lofrumento et al., 2011; Lull and Block, 2010). Post-mortem brains of human PD patients consistently display evidence of inflammation and microglial activation, first shown by McGeer and colleagues (McGeer et al., 1988). Additionally, increased
Conclusion
Because microglial activation and neuroinflammation contribute to the initiation and progression of neurodegeneration in PD, several drugs targeting neuroinflammation have been reported to attenuate the loss of DA neurons and the behavioral deficits seen in animal models of PD. Unfortunately, most have failed in clinical trials. Therefore, there is a significant need to find therapeutics that target the disease process. This study provides evidence that Nav1.6 highly expressed in activated
Funding
This work was supported in part by the following NIH Grants: R01ES021800, R01NS088627, P30ES005022, U01NS079249, P30NS055077 and P50AG025688. Additional support was provided by the Michael J Fox Foundation and private support from the Glenn and Karen Leppo Parkinson's Research Fund, the Richard Nicely Parkinson's Research Fund and the Alan and Janice Woll Parkinson's Research Fund. The content of this article is solely the responsibility of the authors and does not necessarily represent the
Conflict of interest
The authors declare that they have no competing financial interests or other conflicts of interest.
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