Sigma-1 receptor deficiency reduces MPTP-induced parkinsonism and death of dopaminergic neurons

Sigma-1 receptor (σ1R) has been reported to be decreased in nigrostriatal motor system of Parkinson's disease patients. Using heterozygous and homozygous σ1R knockout (σ1R+/− and σ1R−/−) mice, we investigated the influence of σ1R deficiency on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-impaired nigrostriatal motor system. The injection of MPTP for 5 weeks in wild-type mice (MPTP-WT mice), but not in σ1R+/− or σ1R−/− mice (MPTP-σ1R+/− or MPTP-σ1R−/− mice), caused motor deficits and ~40% death of dopaminergic neurons in substantia nigra pars compacta with an elevation of N-methyl-d-aspartate receptor (NMDAr) NR2B phosphorylation. The σ1R antagonist NE100 or the NR2B inhibitor Ro25-6981 could alleviate the motor deficits and the death of dopaminergic neurons in MPTP-WT mice. By contrast, MPTP-σ1R+/− mice treated with the σ1R agonist PRE084 or MPTP-σ1R−/− mice treated with the NMDAr agonist NMDA appeared to have similar motor deficits and loss of dopaminergic neurons as MPTP-WT mice. The pharmacological or genetic inactivation of σ1R suppressed the expression of dopamine transporter (DAT) in substantia nigra, which was corrected by NMDA. The activation of σ1R by PRE084 enhanced the DAT expression in WT mice or σ1R+/− mice. By contrast, the level of vesicular monoamine transporter 2 (VMAT2) in σ1R+/− mice or σ1R−/− mice had no difference from WT mice. Interestingly, MPTP-WT mice showed the reduction in the levels of DAT and VMAT2, but MPTP-σ1R−/− mice did not. The inactivation of σ1R by NE100 could prevent the reduction of VMAT2 in MPTP-WT mice. In addition, the activation of microglia cells in substantia nigra was equally enhanced in MPTP-WT mice and MPTP-σ1R−/− mice. The number of activated astrocytes in MPTP-σ1R−/− mice was less than that in MPTP-WT mice. The findings indicate that the σ1R deficiency through suppressing NMDAr function and DAT expression can reduce MPTP-induced death of dopaminergic neurons and parkinsonism.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor symptoms, including bradykinesia and tremor, and a progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). 1,2 Sigma-1 receptor (σ 1 R), previously named the opioid receptor sigma-1, is found primarily in motoneurons localized in the brainstem and spinal cord. 3 The σ 1 R is expressed in dopaminergic neurons and astrocytes. 4 The σ 1 R agonist PRE084 has been reported to exert neurorestorative effects on 6-hydroxydopamine (6-OHDA)-induced parkinsonism. 4 Using positron emission tomography, the σ 1 R-binding sites are found to be reduced in the brains of early-phase PD patients. 5 However, the influence of σ 1 R deficiency on the pathogenesis of PD has not yet been reported.
Dopamine toxicity is involved in the etiology of PD. 6 The σ 1 R-binding sites on dopaminergic nerve terminals are involved in increasing dopamine release by enhancing N-methyl-D-aspartate receptors (NMDAr). 7 The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolized to 1-methyl-4-phenylpyridinium in glial cells selectively impairs dopaminergic neurons in SNpc through disrupting respiratory enzymes and causing oxidative damage. 8 The dopamine transporter (DAT), a high-affinity transmembrane protein, is responsible for dopamine reuptake from the synaptic cleft and the transportation of 1-methyl-4phenylpyridinium into dopaminergic nerve terminals. 9 The σ 1 R is co-expressed with DAT in dopaminergic neurons. 4 Furthermore, the low density of DAT has been confirmed in the brains of PD patients. 5 The activation of σ 1 R enhances the Ca 2+ influx across NMDAr through increasing the phosphorylation of NR2B or the trafficking NMDAr to the plasma membrane. 10,11 The NMDAr NR2B inhibitor can attenuate MPTP-or 6-OHDAinduced parkinsonian symptoms and neurodegeneration. 12 The σ 1 R deficiency has been demonstrated to reduce Aβinduced neuronal cell death through suppressing NR2B phosphorylation. 13 The inflammation is a predominant aspect of PD, manifested by glial activation with the expression of pro-inflammatory mediators. 14 Sustained neuro-inflammation can exacerbate the degeneration of dopaminergic neurons. 15 The blockade of σ 1 R has been reported to inhibit methamphetamine-induced astrogliosis. 16 Moreover, the 6-OHDA-induced spontaneous rotations or decline of dopaminergic fibers in σ 1 R knockout mice seem to be less than 1 State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China and 2 Department of Physiology, Nanjing Medical University, Nanjing, China *Corresponding author: L Chen, Department of Physiology, Nanjing Medical University, Hanzhong Road 140, Nanjing 210029, China. Tel: +86 025 86862878; Fax: +86 025 86260332; E-mail: lingchen@njmu.edu.cn those in wild-type (WT) mice. 4 Paquette et al. reported that the blockade of σ 1 R could attenuate abnormal involuntary movements induced by 6-OHDA. 17 In this study, we employed heterozygous and homozygous σ 1 R knockout (σ 1 R +/ − and σ 1 R − / − ) mice to investigate the influence of σ 1 R deficiency on MPTP-induced parkinsonism and death of dopaminergic neurons, and the underlying molecular mechanisms. Using the experimental PD models of MPTP-treated σ 1 R +/ − mice and σ 1 R − / − mice, the present study provides in vivo evidence that the σ 1 R deficiency through suppressing NMDAr function and DAT expression can attenuate MPTP-induced dopaminergic neurodegeneration and parkinsonism.
Results σ 1 R deficiency reduces MPTP-induced motor deficits. The locomotion ability and motor coordination were examined on days 3-11 after the last MPTP injection ( Figure 1). In the open-field test (OFT), the traveled distance ( Figure 2a) and the rearing number ( Figure 2b) were not significantly different between WT and σ 1 R − / − mice. In comparison with WT mice, MPTP-treated WT mice (MPTP-WT mice) showed a significant decrease in the traveled distance (Po0.01, n = 12) and the rearing number (Po0.05, n = 12), whereas MPTP- In beam walking test (BWT), the walking time to traverse the beam in σ 1 R − / − mice did not differ significantly from WT mice (P40.05, n = 12; Figure 2c). Notably, the prolongation of walking time to traverse the beam was observed in MPTP-WT mice compared with WT mice (Po0.05, n = 12), but not in MPTP-σ 1 R − / − mice (P40.05, n = 12).

Discussion
Using the MPTP-σ 1 R +/ − and MPTP-σ 1 R − / − mice models, the present study provides evidence that the σ 1 R deficiency can reduce the MPTP-induced parkinsonism and death of dopaminergic neurons. This conclusion is deduced mainly from the following results. The MPTP-induced motor deficits and death of dopaminergic neurons in WT mice were alleviated by the blockade of σ 1 R. By contrast, MPTP failed to induce the motor deficits and the death of dopaminergic neurons in σ 1 R +/ − mice and σ 1 R − / − mice. MPTP-σ 1 R +/ − mice treated with the σ 1 R agonist PRE084 (1.0 mg/kg) or MPTPσ 1 R − / − mice treated with NMDA appeared to have the same motor deficits and death of dopaminergic neurons as MPTP-WT mice. There is, however, an apparently conflicting report describing that the administration of PRE084 at the dose of 0.3 mg/kg, but not the dose of 1.0 mg/kg, can improve the spontaneous forelimb use and reduce the death of dopaminergic neurons in mice subjected to 6-OHDA lesion. 4 The behavioral, biochemical or electrophysiological effects of σ 1 R agonists show a biphasic, inverted U (bell) shape and dose-dependent. 20 Thus, this discord may arise from the difference in the experimental models or the dose of PRE084 used. Influence of σ 1 R deficiency-reduced NMDAr activation on motor behaviors. The first question we should address may be whether the σ 1 R deficiency affects the motor behaviors. Using OFT and BWT, we found that pharmacological or genetic inactivation of σ 1 R failed to affect the locomotion ability and motor coordination, which are in agreement with previous studies. 21,22 In addition, σ 1 R − / − mice did not appear the spontaneous turning behavior and forelimb use asymmetry as assessed by the cylinder test and the stepping test. 4 The stride length in the footprint test that is a sensitive assay for motor coordination shows no difference between WT mice and σ 1 R − / − mice and the swimming speed in the swimming test in σ 1 R − / − mice is faster than controls. 3 The time stayed on the rotated rod with constant speeds (4-36 r.p.m.) in σ 1 R +/ − and σ 1 R − / − mice had no significant difference from WT mice. There are, however, conflicting reports describing the motor coordination defects in σ 1 R − / − mice that is detected by accelerating RT with a rate of 4-50 r.p.m. for 2.5 min. 3,23 The contradictory results may arise from the difference in the protocol of RT, because we observed that σ 1 R − / − mice, but not σ 1 R +/ − mice, showed a short latency on the accelerating rotarod in comparison with WT mice (data not shown). Monville et al. reported that the incremental constant protocol in RT is more sensitive to detect the presence of lesion, whereas the accelerating protocol provides a more discriminative test to correlate motor deficits. 24 Rustay et al. found that both accelerating and fixed-speed rotarod performance can vary under different condition. 25 On the other hand, the NMDAr is essential for mediating excitatory transmission at corticostriatal dopaminergic synapses. The activation of σ 1 R increases both dopamine and glutamate release. 7,26 In addition to degeneration of dopaminergic neurons, abnormal function of striatal NMDAr has been implicated in the development of motor deficits. 27 Recently, Xiu et al. have reported that the chronic treatment with the NMDAr antagonist MK801 does not affect the coordination or motor function on accelerating rotarod. 28 Thus, it is conceivable that the σ 1 R deficiency-reduced NMDAr function does not produce the motor deficits.
σ 1 R deficiency suppresses NMDAr to prevent MPTP neurotoxicity. Glutamate toxicity has been noted as a main source of the MPTP-impaired dopaminergic system. 29 The tyrosine phosphorylation of NR2B is enhanced by the application of 6-OHDA. 30 The blockade or the knockout of σ 1 R can reduce the levels of phospho-NR2B in substantia nigra or hippocampus. 31 More importantly, the MPTPinduced increase of phospho-NR2B was attenuated by the blockade or the knockout of σ 1 R. One earlier study has reported the MPTP binding to σ protein in C57BL/6 mouse brain membranes. 32 Thus, further studies are needed to evaluate whether MPTP via the activation of σ 1 R enhances σ 1 R deficiency reduces DAT to prevent MPTP neurotoxicity. An important finding in this study is that the pharmacological or genetic inactivation of σ 1 R reduced the DAT expression in dopaminergic neurons, but failed to affect the VMAT2 level. The compensatory downregulation of DAT in early PD is thought to maintain dopamine levels in the synapse. 35 Zhang and Li have recently reported that DAT expression depends on the striatal extracellular dopamine concentration. 36 The activation of σ 1 R through enhancing NMDAr can increase the dopamine release. 7 Indeed, the application of NMDA in σ 1 R − / − mice could enhance the expression of DAT. Therefore, one possible explanation is that the σ 1 R deficiency reduces the release of dopamine via the suppression of NMDAr leading to the compensatory decline in the expression of DAT. On the other hand, MPTP-WT mice appeared the reduction of DAT and VMAT2 levels, which could be rescued by the blockade or knockout of σ 1 R. An earlier study reported that the MPTP-induced loss of dopaminergic neurons was accompanied by a decline in DAT and VMAT2 levels. 37 Therefore, it is suggested that the reduction of DAT and VMAT2 in MPTP-WT mice arises from the loss of dopaminergic neurons. In vivo studies have shown that the knockout or the inhibition of DAT can prevent MPTPinduced neurotoxicity. 38,39 The DAT-overexpressing mice are highly sensitive to MPTP neurotoxicity. 9 The increased dopamine uptake is able to elevate the unique vulnerability of dopamine neurons in PD. Thus, it is possible that the σ 1 R deficiency through downregulating DAT expression can reduce the MPTP-impaired dopaminergic neurons.
σ 1 R deficiency suppresses MPTP-induced astrocyte activation. Consistent with an earlier study in the mouse model, 40 the injection of MPTP could stimulate the activation of astrocytes or microglia cells. In mice of intrastriatal 6-OHDA lesions, the microglia cell activation contributes to the neurodegenerative process during the rapid phase of dopamine cell death, and persists for weeks. 41 The σ 1 R is expressed in astrocytes and microglia cells. 42 The 6-OHDAinduced microglia cell activation can be attenuated by the σ 1 R agonist PRE084. 4 The activation of σ 1 R has the antiinflammatory effects in ALS mice. 43 However, our results showed that the σ 1 R deficiency had no effect on MPTPinduced activation of microglia cells. By contrast, the number of activated astrocyte in MPTP-σ 1 R − / − mice was less than that in MPTP-WT mice. In addition, the blockade of σ 1 R by NE100 in MPTP-WT mice could reduce the activated astrocyte. Recently, a σ 1 R antagonist has been reported to attenuate methamphetamine-induced neurotoxicity and astrogliosis through a blockade of oncostatin M receptor/ gp130 signaling and STAT3 phosphorylation. 16,44 Therefore, the reduced astrocyte activation in MPTP-σ 1 R − / − mice is thought to have two possibilities: one is that the σ 1 R deficiency reduces the MPTP-induced neuronal damage and the other is that the σ 1 R deficiency can suppress the astrocyte activation. Further work will be required to determine these possibilities. The decreased σ 1 R-binding site in early Alzheimer's disease is reported to have a protective effect against Alzheimer's disease susceptibility in a Japanese population. 45 By contrast, the high expression of σ 1 R with the APOE ε4 allele in Chinese or Australian populations has been reported to advance cognitive dysfunction and pathologic stages of Alzheimer's disease. 46 Despite the association between the reduction of σ 1 R and the early PD has not been reported, the present study suggests that the σ 1 R deficiency in the mice model of MPTP-induced PD might exert the neuroprotection effects, which would open new doors for preventing and treating PD.

Materials and Methods
Animals. All animal handling procedures followed the Guidelines for Laboratory Animal Research of Nanjing Medical University. The use of animals was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. As a σ 1 R knockout line, we chose the well-characterized Oprs1 mutant (+/ − ) Oprs1Gt(IRESBetageo)33Lex litters on a C57BL/6 J × 129 S/SvEv mixed background, 21 which we obtained from the Mutant Mouse Resource Regional Centre at the University of California, Davis. The genotype of mice was identified by PCR using genomic DNA from tail biopsies (Supplementary Figure 1A) with the following primers sequences: (a) 5′-TCTGAGTACGTGCTGCTCTTCG-3′; (b) 5′-AT AAACCCTCTTGCAGTTGCATC-3′; (c) 5′-GAAACTGCCGTGTTCTGTTTCC-3′, and PCR reaction parameters: 30 cycles of 94°C (15 s), 55°C (30 s) and 72°C (40 s). 21 The western blotting analysis showed the reduction or lack of σ 1 R protein level in σ 1 R +/ − mice and σ 1 R − / − mice, respectively (Supplementary Figure 1B). The mice were maintained under constant environmental conditions (temperature 23 ± 2°C, humidity 55 ± 5% and 12 : 12-h light/dark cycle) in the Animal Research Center of Nanjing Medical University with free access to food and water. Consistent with the report by Sabino et al., 21 the development of σ 1 R +/ − mice and σ 1 R − / − mice appeared grossly normal.
Experimental design. Male 12-week-old WT mice (25.26 ± 0.84 g), σ 1 R +/ − mice (25.19 ± 0.91 g) and σ 1 R − / − mice (25.35 ± 0.76 g) were used at the beginning of all experiments. A total of 176 WT mice, 80 σ 1 R +/ − mice and 120 σ 1 R − / − mice were divided into five experimental groups to examine (a) influence of σ 1 R deficiency on MPTP-impaired motor behaviors (n = 12) and MPTP neurotoxicity (n = 8); (b) effects of σ 1 R antagonist and agonist on MPTP-impaired motor behaviors (n = 12) and MPTP neurotoxicity (n = 8); (b) influence of σ 1 R deficiency on MPTP-increased NR2B phosphorylation (n = 8) and NMDAr-mediated MPTP neurotoxicity (n = 8); (d-e) influence of σ 1 R deficiency on DAT and VAMT2 expression (n = 8) and MPTP-induced inflammatory (n = 8). Behavioral tests were carried out starting from day 3 after the last injection of MPTP or drugs to evaluate spontaneous locomotion and motor coordination. At the end of the behavioral tests, the mice were perfusion-fixed for histological examination (time chart of experimental procedure; Figure 1). In addition, on day 3 after the last injection of MPTP, the mice were decapitated for either biochemical assays or western blotting.
Behavioral examination. Three different behavioral tests were carried out (9000-1400) under following sequence: OFT → BWT → RT. Tests were spaced by 24 h. These behavioral tests were recorded by a video monitor (Winfast PVR; Leadtek Research Inc., Fremont, CA, USA). The results of OFT and BWT were analyzed using TopScan Lite 2.0 (Clever Sys, Reston, VA, USA) and the results of RT were analyzed by Rota-Rod microprocessor 47600 (Ugo Basile, Biological Research Apparatus, Varese, Italy).
Open-field test: Each mouse was placed in a clear, open-top, square Plexiglas box (30 × 30 × 40 cm 3 ) in a subdued room and allowed to freely explore for 6 min. Rearing number and traveled distance were measured within 6 min. 48 Beam walking test: The challenging beam was a 1-m long wooden beam suspended 23 cm above a bench top, which was covered with soft pads to protect the mouse in case of a fall. The beam was divided in four gradually narrowing sections (25 cm/section) leading to the mouse's home cage. The beam widths of the four sections were 3.5, 2.5, 1.5 and 0.5 cm in decreasing order. The beam was covered with surgical tape that provided sufficient surface traction for the animals to walk on. There were 1-cm-wide ledges hanging 1 cm below each side of the beam to encourage the mice to use their normal gait strategies even when their limbs slipped. All mice were pre-trained for 2 consecutive days on traversing the beam. On the third day, each mouse was given five trials (inter-trial intervals = 10-12 s), and the average time was calculated. 49 Rotarod test: The rotarod apparatus (Ugo Basile) was used to measure forelimb and hindlimb motor coordination and balance. Two different protocols are widely used, incremental fixed speeds or an accelerating protocol. The former may be most appropriate to detect the presence of a lesion, whereas the latter is to be preferred to characterize the magnitude and extent of depletion in different animals. 24 The first protocol was selected in this study to examine MPTP-induced parkinsonism. Mice were placed on the rotating rod using the following steps: on days 1 and 2, mice learned to stay on the rotarod at constant speed (20 r.p.m.) for 300 s. At day 3, motor coordination was assessed on the rotarod with five constant speeds (4-36 r.p.m.) for a maximum of 60 s at each speed. For each trial, the time until the mice fall off the rod was recorded. The animals were tested two times at each speed with a rest of 20 min between each trial. 24 Histological examination and quantitative analyses. Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.), and then perfused with 4% paraformaldehyde. Coronal sections (30 μm) were cut using a cryostat. The immunostaining of TH, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1) was performed using the following primary antibodies: chicken anti-TH (1 : 1000; Abcam, Cambridge, UK), rabbit anti-GFAP (1 : 1000; Cell Signaling Technology, Inc., Boston, MA, USA) and goat anti-Iba1 (1 : 1000; Abcam) at 4°C overnight. Then the sections were incubated in biotin-labeled goat anti-chicken IgG antibody (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-rabbit IgG antibody (1 : 400; Santa Cruz) or rabbit anti-goat antibody (1 : 400; Santa Cruz). Immunoreactivity was visualized by the avidin-biotin-horseradish peroxidase complex (ABC Elite; Vector Laboratories, Inc., Burlingame, CA, USA). For DAT immunostaining, the brains were processed for paraffin embedding. Coronal paraffin sections (5 μm) were incubated in rabbit anti-DAT antibody (1 : 200; Alomone Labs, Jerusalem, Israel) at 4°C overnight. The sections were incubated in Cy3conjugated goat anti-rabbit antibody (1 : 200, Jackson ImmumoResearch Lab., West Grove, PA, USA) for 2 h. For Hoechst staining, the paraffin sections (5 μm) were incubated in Hoechst 33342 (1 μg/ml; Cell Signaling Technology) for 2 min. The DATpositive cells and Hoechst-positive cells were observed using a fluorescent light microscope (Olympus, DP70; Tokyo, Japan).
The number of TH-positive cell bodies in SNpc (12 sections per mouse) was determined by Microbrightfield Stereo Investigator software according to the optical fractionator method (Microbrightfield, Williston, VT, USA). 50 The total number of THpositive cells was estimated using the optical fractionator formula: number of neurons = 1/ssf (slice sampling fraction) × 1/asf (area sampling fraction) × 1/tsf (thickness sampling fraction) × Σ (number of objects counted). The Iba1-and GFAPpositive cells in substantia nigra (12 sections per mouse) were counted in an area of 500 × 500 μm 2 per section by a conventional light microscope (Olympus, DP70, × 40) with an x-y motorized stage controlled by the New CAST software (Visiopharm, Hørsholm, Denmark). The number of Iba1-and GFAP-positive cells was normalized by value of WT mice.