A previous report demonstrated that DA
A previous report demonstrated that DA-dependent oxidative stress may be the initial event in MA neurotoxicity (Cubells et al., 1994). Oxidative burdens at early stage might be a prerequisite for neurotoxic scenarios induced by 3-FMA, and they are in line with MA case (Shin et al., 2012, Shin et al., 2014, Shin et al., 2017a). DA rapidly auto-oxidizes to form reactive quinone derivatives, hydrogen peroxide and free radicals when MA redistributes DA from synaptic vesicles to 4-DAMP synthesis (Graham et al., 1978, Slivka and Cohen, 1985, Cubells et al., 1994, Acikgoz et al., 1998). Thus, it is plausible that DA-dependent oxidative stress may contribute to the specific neurotoxicity induced by 3-FMA or MA. Importantly, Granado et al. (2011b) demonstrated that nuclear factor-erythroid 2-related factor 2 (Nrf2) knockout mice exacerbated MA-induced oxidative stress, and damage to dopamine neurons in the striatum, suggesting that the protective role of Nrf2 in MA-induced neurotoxicity. A possible role of Nrf2 in 3-FMA-induced neurotoxicity remains to be determined. Neither 3-FMA nor MA produced nigral loss (Supplementary Fig. S3) or GFAP-labeled astrogliosis in the striatum (Supplementary Fig. S4). Current result is in line with our previous report that a single, high dose of MA does not significantly alter TH-immunoreactivity in the substantia nigra as well as GFAP-immunoreactivity in the striatum (Dang et al., 2017b). However, Iba-1-labeled microgliosis in the striatum is specific for the neurotoxicity induced by 3-FMA or MA, although precise mechanism remains elusive. A strong support for a dual role of microglia may be due to distinct microglial phenotypes, which have been broadly categorized into M1 (classically activated, pro-inflammatory) and M2 (alternatively activated, anti-inflammatory) (Martinez and Gordon, 2014, Franco and Fernandez-Suarez, 2015, Kawabori and Yenari, 2015, Thompson and Tsirka, 2017). We observed that 3-FMA or MA administration significantly increased mRNA expression of M1 phenotypic markers (CD16, CD32, and CD86) and did not significantly change mRNA expression of M2 phenotypic markers (Arginase 1 and CD206), suggesting that microgliosis induced by 3-FMA and MA treatment leads to neuroinflammation. In this study, dopamine D1 receptor antagonism to 3-FMA insult by SCH23390 up-regulated M2 phenotype (i.e., Arginase 1 and CD206 mRNA expressions). However, both SCH23390 and sulpiride did not affect M2 phenotype mRNA levels in MA insult. Although this discrepancy remains to be fully elucidated, we cannot rule out the possibility that a fluorine atom at the 3 position of phenyl group in the chemical structure of 3-FMA may produce this different profile of toxicity as compared to MA. MA treatment up-regulates pro-apoptotic protein (i.e., Bax) and down-regulates anti-apoptotic proteins (i.e., Bcl-2, Bcl-xl) in the brain, which consequently induce caspase activation (Jayanthi et al., 2001, Nguyen et al., 2015, Dang et al., 2017a, Timucin and Basaga, 2017). It has been reported that caspase activation is involved in the induction of TUNEL-positive cells via apoptotic processes after a single, high dose of MA in the striatum of mice (Dang et al., 2016, Dang et al., 2017a, Shin et al., 2017a). Importantly, compelling evidence indicated that caspase activation regulates microglial activation via a PKCδ-dependent pathway (Burguillos et al., 2011). Indeed, our previous reports demonstrated that PKCδ mediates dopaminergic degeneration in neuroinflammation induced by MA (Shin et al., 2011, Shin et al., 2012, Shin et al., 2014, Dang et al., 2015) or para-methoxymethamphetamine (PMMA) (Shin et al., 2016). The combination of oxidative stress and microglial activation generates a vicious cycle, that appears to lead to a progressive neuronal apoptosis (Hald and Lotharius, 2005, Shin et al., 2012, Shin et al., 2014, Dang et al., 2016). We propose that oxidative stress, microglial activation and pro-apoptotic signaling might be overlapping neurotoxic outcomes in 3-FMA and MA, although underlying mechanism of 3-FMA on dopaminergic receptor modulation is different from that of MA.