Preceded GFAP upregulation (Fig. 4 C I). Although reduced expression of LIF and IL-6 is likely not associated with reduced GRP78 or CHOP expression in ATF6a 2/2 mice, these results suggest that ATF6a may transcriptionally regulate the expression of astrogliosis-inducing factors after MPTP/P injection. Consistent with the immunohistochemical results, GLT-1 expression was not significantly different between wild-type and ATF6a 2/2 mice in the control condition or after MPTP/P injection (Fig. S2 D).dopaminergic neurons and GFAP-positive astrocytes without reducing the number of TH-positive neurons or the intensity of TH. RT-PCR analyses revealed enhanced activation of ATFa and PERK/ATF4 pathways, but not of the Ire1/XBP1 pathway, in IN19-administrated mice (Fig. 5 B I, II, III). Unlike MPTP/P administration, IN19 administration did not upregulate GFAP or Iba1 (Fig. 5 B IV), suggesting that the JW-74 effect of IN19 on UPR was not mediated by general neuronal damage. IN19 also enhanced eIF2a phosphorylation in dopaminergic neurons (Fig. S3 B), as 60940-34-3 web previously described [11]. Next, we assessed the neuroprotective property of IN19 after 
MPTP/P injections. When mice were given IN19 (10 mg/kg, p.o. in saline, including 10 Cremophore EL and 10 DMSO) 24 h and 2 h before MPTP/P injection, the number of TH-positive neurons in the SN and the intensity of TH in the CPu were significantly increased (Fig. 6 A). Consistently, the number of activated caspase 3-positive, TH-positive neurons (Fig. 6 B) decreased in the SN, and expression of BDNF in the CPu increased in the astrocytes of mice given IN19 after MPTP/P injection (Fig. 6 C I, II). Importantly, expression of GFAP in the CPu also mildly, but significantly, increased in mice given IN19 after MPTP/P injection (Fig. 6 C I, II), suggesting that IN19 may protect dopaminergic neurons, at least in part, through the activated astrocytes after MPTP/P administration.DiscussionIn this study, we first demonstrated the activation of the UPR in a chronic MPTP/P injection model. Of the 3 UPR branches, the ATF6a and PERK/eIF2a/ATF4 pathways were preferentially activated after MPTP/P injections (Fig. 1 B). We also observed a trend that the PERK/eIF2a/ATF4 pathway was highly activated after the 1st MPTP/P injection (8 h after injection; Fig. 1 B II), but the ATF6 pathway was activated for longer periods over the course of the MPTP/P injections (1st through 5th injections; Fig. 1 B I). These results are consistent with those of previous reports demonstrating differential activation between the 3 UPR branches after PD-related stresses caused by MPP+ or 6OHDA in cultured cells [9,19]. Taken together with a recent report, which demonstrated a direct link after MPP+ treatment between p38 MAP kinase and ATF6a [12], these findings suggest critical roles for the ATF6a and PERK/eIF2a/ATF4 pathways as defense systems against PD-related neurotoxins. Analyses of wild-type and ATF6a 2/2 mice showed accelerated degeneration of the nigrostriatal neurons in ATF6a 2/2 mice (Fig. 2 A I, II, III) after the earlier MPTP/P injections (2nd 16574785 and 3rd injections), but not after the later injections (6th through10th injections). Similarly, Ub accumulation was observed in ATF6a 2/2 dopaminergic neurons after the early MPTP/P injections (2nd and 3rd injections; Fig. 2 B I). However, Ub-positive inclusions, which were abundantly observed in ATF6a 2/2 
mice after acute MPTP injection [12], were observed only in 29 of ATF6a 2/2 mice after the last inje.Preceded GFAP upregulation (Fig. 4 C I). Although reduced expression of LIF and IL-6 is likely not associated with reduced GRP78 or CHOP expression in ATF6a 2/2 mice, these results suggest that ATF6a may transcriptionally regulate the expression of astrogliosis-inducing factors after MPTP/P injection. Consistent with the immunohistochemical results, GLT-1 expression was not significantly different between wild-type and ATF6a 2/2 mice in the control condition or after MPTP/P injection (Fig. S2 D).dopaminergic neurons and GFAP-positive astrocytes without reducing the number of TH-positive neurons or the intensity of TH. RT-PCR analyses revealed enhanced activation of ATFa and PERK/ATF4 pathways, but not of the Ire1/XBP1 pathway, in IN19-administrated mice (Fig. 5 B I, II, III). Unlike MPTP/P administration, IN19 administration did not upregulate GFAP or Iba1 (Fig. 5 B IV), suggesting that the effect of IN19 on UPR was not mediated by general neuronal damage. IN19 also enhanced eIF2a phosphorylation in dopaminergic neurons (Fig. S3 B), as previously described [11]. Next, we assessed the neuroprotective property of IN19 after MPTP/P injections. When mice were given IN19 (10 mg/kg, p.o. in saline, including 10 Cremophore EL and 10 DMSO) 24 h and 2 h before MPTP/P injection, the number of TH-positive neurons in the SN and the intensity of TH in the CPu were significantly increased (Fig. 6 A). Consistently, the number of activated caspase 3-positive, TH-positive neurons (Fig. 6 B) decreased in the SN, and expression of BDNF in the CPu increased in the astrocytes of mice given IN19 after MPTP/P injection (Fig. 6 C I, II). Importantly, expression of GFAP in the CPu also mildly, but significantly, increased in mice given IN19 after MPTP/P injection (Fig. 6 C I, II), suggesting that IN19 may protect dopaminergic neurons, at least in part, through the activated astrocytes after MPTP/P administration.DiscussionIn this study, we first demonstrated the activation of the UPR in a chronic MPTP/P injection model. Of the 3 UPR branches, the ATF6a and PERK/eIF2a/ATF4 pathways were preferentially activated after MPTP/P injections (Fig. 1 B). We also observed a trend that the PERK/eIF2a/ATF4 pathway was highly activated after the 1st MPTP/P injection (8 h after injection; Fig. 1 B II), but the ATF6 pathway was activated for longer periods over the course of the MPTP/P injections (1st through 5th injections; Fig. 1 B I). These results are consistent with those of previous reports demonstrating differential activation between the 3 UPR branches after PD-related stresses caused by MPP+ or 6OHDA in cultured cells [9,19]. Taken together with a recent report, which demonstrated a direct link after MPP+ treatment between p38 MAP kinase and ATF6a [12], these findings suggest critical roles for the ATF6a and PERK/eIF2a/ATF4 pathways as defense systems against PD-related neurotoxins. Analyses of wild-type and ATF6a 2/2 mice showed accelerated degeneration of the nigrostriatal neurons in ATF6a 2/2 mice (Fig. 2 A I, II, III) after the earlier MPTP/P injections (2nd 16574785 and 3rd injections), but not after the later injections (6th through10th injections). Similarly, Ub accumulation was observed in ATF6a 2/2 dopaminergic neurons after the early MPTP/P injections (2nd and 3rd injections; Fig. 2 B I). However, Ub-positive inclusions, which were abundantly observed in ATF6a 2/2 mice after acute MPTP injection [12], were observed only in 29 of ATF6a 2/2 mice after the last inje.