Intracellular Hydrogen Peroxide Produced by 6-Hydroxydopamine is a Trigger for Nigral Dopaminergic Degeneration Via Rapid Inux of Extracellular Zn2+

To elucidate the mechanism of 6-hydroxydopamine (6-OHDA)-induced Zn 2+ toxicity, which is involved in neurodegeneration in the substantia nigra pars compacta (SNpc) of rats, we postulated that intracellular hydrogen peroxide (H 2 O 2 ) produced by 6-OHDA is a trigger for intracellular Zn 2+ dysregulation in the SNpc. Intracellular H 2 O 2 level in the SNpc elevated by 6-OHDA was completely inhibited by co-injection of GBR 13069 dihydrochloride (GBR), a dopamine reuptake inhibitor, suggesting that 6-OHDA taken up through dopamine transporters produces H 2 O 2 in the intercellular compartment of dopaminergic neurons. When the SNpc was perfused with H 2 O 2 , H 2 O 2 accumulated glutamate in the extracellular compartment and the accumulation was inhibited in the presence of N-(p-amylcinnamoyl)anthranilic acid (ACA), a blocker of the transient receptor potential melastatin 2 (TRPM2) channels. In addition to 6-OHDA, H 2 O 2 also induced intracellular Zn 2+ dysregulation via AMPA receptor activation followed by nigral dopaminergic degeneration. Furthermore, 6-OHDA-induced nigral dopaminergic degeneration was completely inhibited by co-injection of HYDROP, an intracellular H 2 O 2 scavenger or GBR into the SNpc. The present study indicates that H 2 O 2 is produced by 6-OHDA taken up through dopamine transporters in the SNpc, is retrogradely transported to presynaptic glutamatergic terminals, activates TRPM2 channels, accumulates glutamate in the extracellular compartment, and induces intracellular Zn 2+ dysregulation via AMPA receptor activation, resulting in nigral dopaminergic degeneration. It is likely that intracellular H 2 O 2 , but not extracellular H 2 O 2 , is a key trigger for nigral dopaminergic degeneration via intracellular Zn 2+ dysregulation.


Introduction
The substantia nigra pars compacta (SNpc), a mesencephalic nucleus of the basal ganglia, serves in the regulation of voluntary movement. In Parkinson's disease (PD), dopaminergic neurons are selectively damaged and degenerated, resulting in progressive motor dysfunction symptoms, e.g., resting tremor, bradykinesia, rigidity, and postural imbalance [1]. Most PD patients are sporadic and aging is the major risk factor [2,3]. The exact mechanism of PD pathogenesis remains unclear, while oxidative stress hypothesis is well known as the pathogenetic mechanism. The SNpc is enriched with dopamine, which can undergo both enzymatic oxidation via monoamine oxidase and nonenzymatic autoxidation, resulting in the generation of hydrogen peroxide (H 2 O 2 ) and oxyradicals (superoxide anion radical and hydroxyl radical) in the SNpc [4].
Among intracellular ROS derived from 6-OHDA and PQ, H 2 O 2 readily passes through cell membranes through aquaporin channels [10,11] and the elevation in the extracellular compartment can excites glutamatergic neuron terminals via TRPM2 channel activation [12][13][14], which innervates dopaminergic neurons in the SNpc. To elucidate the mechanism of 6-OHDA-induced Zn 2+ toxicity, which is involved in dopaminergic degeneration in the SNpc of rats, we postulated that intracellular H 2 O 2 produced by 6-OHDA is a trigger for intracellular Zn 2+ dysregulation in the SNpc. We examined an idea that 6-OHDA taken up through dopamine transporters produces H 2 O 2 and that toxic signal of H 2 O 2 is readily converted into that of Zn 2+ .

Animals and chemicals
Wistar rats (male, 10-20 weeks of age) were obtained from Japan SLC (Hamamatsu, Japan). They were housed under the standard laboratory conditions (23 ± 1°C, 55 ± 5% humidity) and had access to water and food ad libitum. The present experiments were done in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka, which refer to American Association for Laboratory Animals Science and the guidelines laid down by the NIH (NIH Guide for the Care and Use of Laboratory Animals) in the USA. All experimental protocols were approved by the ethics committee of the University of Shizuoka.
ZnAF-2DA, a membrane-permeable Zn 2+ uorescence probe (Sekisui Medical Co., LTD, Hachimantai, Japan) is readily taken up into the cells through the cell membrane and is hydrolyzed by esterase in the cytosolic compartment, resulting in generation of ZnAF-2 [15]. Intracellular ZnAF-2, which cannot permeate the cell membrane, is selectively bound to Zn 2+ , but not bound to other divalent cations such as
Tyrosine Hydroxylase (Th) Immunostaining Two weeks after the surgical operation for the treatment with 6-OHDA, the rats were anesthetized and perfused with ice-cold 4% paraformaldehyde in PBS. The brain was excised from the rats followed by overnight xation in 4% paraformaldehyde in PBS at 4°C. Fixed brains were cryopreserved in 30% sucrose in PBS for 2 day and frozen in Tissue-Tek Optimal Cutting Temperature embedding medium. Coronal slices (30 µm) of the brains were prepared in a cryostat at -20°C, picked up on slides, and adhered for 30 min at room temperature. For TH immunostaining, the slides were incubated in blocking solution (3% BSA, 0.1% Triton X-100 in PBS) for 1 h and rinsed with PBS for 5 min followed by overnight incubating in anti-tyrosine hydroxylase antibody (Abcam) at 4°C. The slides were rinsed with PBS for 5 min and incubated in blocking buffer containing Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher) for 3 h at room temperature. The slides were bathed in PBS for 5 min six times, mounted with Prolong Gold antifade reagent, and placed at 4°C or 24 h. Alexa Fluor 633 uorescence was observed in the SNpc using a confocal laser-scanning microscopic system.

In Vitro Dynamics Of Intracellular Zn
Rats were anesthetized with chloral hydrate (400 mg/kg) and decapitated. The brain was quickly excised and bathed in ice-cold choline-Ringer containing 124 mM choline chloride, 2.5 mM KCl, 2.5 mM MgCl 2 , 1.25 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , 26 mM NaHCO 3 , and 10 mM glucose (pH 7.3) to inhibit excessive neuronal excitation. Horizontal slices (400 µm) of the brains were prepared in an ice-cold choline-Ringer solution in a vibratome ZERO-1 (Dosaka Kyoto, Japan) and then bathed in an ice-cold choline-Ringer solution. All solutions used in the experiments were continuously bubbled with 95% O 2 and 5% CO 2 .
Brain slices were then immersed in 10 µM ZnAF-2DA in Ringer solution for 30 min, rinsed in choline-Ringer solution for 20 min, placed in a chamber lled with 800 µM 6-OHDA or 800 µM 6-OHDA + 10 µM CNQX in Ringer solution containing 10 nM ZnCl 2 for 10 min, rinsed in choline-Ringer solution for 15 min, and transferred to a recording chamber lled with Ringer solution. Intracellular ZnAF-2 uorescence (laser, 488.4 nm; emission, 500-550 nm) was observed in the SNpc with a confocal laser-scanning microscopic system. In another experiment, brain slices loaded with ZnAF-2DA were placed in a chamber lled with 800 µM H 2 O 2 or 800 µM H 2 O 2 + 10 µM CNQX in Ringer solution containing 10 nM ZnCl 2 for 10 min and treated in the same manner.

In Vivo Imaging Of Intracellular Ho
The rats anesthetized with chloral hydrate were treated as described above. Injection cannulae (internal diameter, 0.15 mm; outer diameter, 0.35 mm) were carefully and slowly inserted into the both sides of the SNpc (5.3 mm posterior to the bregma, 2.0 mm lateral, 7.0 mm inferior to the dura) to prevent cellular damages. Thirty minutes after the surgical operation, 800 µM 6-OHDA or 800 µM 6-OHDA + 2 µM GBR in saline containing 0.1% ascorbic acid and HYDROP™ (50 µM), were bilaterally injected into the SNpc via cannulae at the rate of 0.2 µl/min for 5 min. Ten minutes later, the injection cannulae were slowly moved from the brain in approximately 3 min and the rats were decapitated. The brain was quickly excised from the rats and brain slices were prepared in the same manner. The brain slices were transferred to a recording chamber lled with Ringer solution. Intracellular HYDROP uorescence was observed in the SNpc with a confocal laser-scanning microscopic system.

In Vivo Microdialysis
The rats anesthetized with chloral hydrate were individually placed in a stereotaxic apparatus in the same manner. The skull was exposed, a burr hole was drilled, and a microdialysis probe (1-mm membrane, Eicom, Kyoto) was carefully and slowly inserted into the right SNpc

Data analysis
The differences between treatments were assessed by one-way ANOVA followed by post hoc testing using the Tukey's test (the statistical software, GraphPad Prism 5). A value of p < 0.05 was considered signi cant. Statistical analysis of the data (means ± standard error) is described in each gure legend.

Results
Intracellular Zn 2+ dysregulation induced by 6-OHDA causes neurodegeneration We examined whether 6-OHDA taken up through dopamine transporters produces H 2 O 2 , which is retrogradely transported to presynaptic TRPM2 channels on glutamatergic terminals through the plasma membranes of dopaminergic neurons in the SNpc and accumulates glutamate in the extracellular compartment via TRPM2 channel activation followed by intracellular Zn 2+ toxicity. When brain slices were bathed in 6-OHDA, intracellular H 2 O 2 level was rapidly elevated in the SNpc and the elevation was completely inhibited by GBR (Fig. 3), suggesting that 6-OHDA taken up through dopamine transporters produces H 2 O 2 in the intercellular compartment of dopaminergic neurons.  (Fig. 4). When brain slices were incubated for 10 min with H 2 O 2 in place of 6-OHDA, H 2 O 2 also rapidly increased intracellular Zn 2+ in the SNpc. The increase in intracellular Zn 2+ was preferentially observed in the SNpc, while the increase was blocked in the presence CNQX (Fig. 5). We did not determine extracellular glutamate concentration during perfusion with 6-OHDA because the presence of 6-OHDA in the perfusate disturbs the measurement [18].
Is nigral dopaminergic degeneration linked with retrograde transport to glutamatergic terminals of intracellular H 2 O 2 produced by 6-OHDA?
Nigral dopaminergic degeneration induced by H 2 O 2 in place of 6-OHDA was determined by TH immunostaining. Staining intensity was also signi cantly reduced in the ipsilateral SNpc, while the reduction was completely rescued by co-injection of ACA (Fig. 6), suggesting the involvement of H 2 O 2sensitive TRPM2 channel activation in neurodegeneration.
On the other hand, 6-OHDA-induced dopaminergic degeneration was completely rescued by co-injection of an intracellular H 2 O 2 scavenger (HYDROP) or a dopamine reuptake inhibitor (GBR) (Fig. 7), suggesting involvement of 6-OHDA-mediated H 2 O 2 production in the intracellular compartment in neurodegeneration.

Discussion
Intracellular Zn 2+ dysregulation induced by 6-OHDA is linked with dopaminergic degeneration in the SNpc [18]. However, the mechanism of the rapid Zn 2+ dysregulation is unknown and it is not proven whether the rapid Zn 2+ dysregulation in the intracellular compartment is a trigger for 6-OHDA-induced PD pathogenesis in rats. We postulated that 6-OHDA-induced production of intracellular H 2 O 2 is closely linked with intracellular Zn 2+ dysregulation in the SNpc, which may lead to 6-OHDA-induced PD pathogenesis in rats. To test the postulation, in the present study, we used a low dose of 6-OHDA, which did not cause any movement disorder in response to apomorphine. 6-OHDA signi cantly caused nigral dopaminergic degeneration, while the neurodegeneration was completely rescued by co-injection of ZnAF-2DA, as an intracellular Zn 2+ chelator, as well as CaEDTA and CNQX, in comparison with the partial rescue effect of ZnAF-2DA on the high dose of 6-OHDA reported previously [18], indicating that 6-OHDAinduced intracellular Zn 2+ dysregulation via AMPA receptor activation causes nigral dopaminergic degeneration. It has been reported that Ca 2+ -and Zn 2+ -permeable GluR2-lacking AMPA receptor activation in the SNpc is involved in age-related vulnerability of nigral dopaminergic neurons [19].
6-OHDA is readily oxidized and generates a number of ROS, e.g., the superoxide anion radical, the hydroxy radical, singlet oxygen, and H 2 O 2 in both extracellular and intracellular compartments [7]. We postulated that presynaptic glutamatergic excitation, which innervates nigral dopaminergic neurons, is required for the rapid intracellular Zn 2+ dysregulation followed by preferential dopaminergic degeneration in the SNpc.
We focused on membrane-permeable H 2 O 2 to prove the postulation, which is an idea that H 2 O 2 is produced by 6-OHDA taken up through dopamine transporters, retrogradely transported to presynaptic TRPM2 channels from postsynaptic dopaminergic neurons, and activates glutamatergic synapses in the SNpc (Fig. 8) neuron-speci c TRPM2 cation channels may contribute to the pathophysiology of neurological disorders, e.g., cerebral ischemia and oxygen-glucose deprivation [14,[24][25][26][27]. The increase in TRPM2 channel expression enhances the susceptibility to ROS-induced cell death in human neuroblastoma SH-SY5Y cells, a dopaminergic neuronal cell line [28]. The expression of TRPM2 cation channels is increased in substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and also PD patients [29].
Furthermore, 6-OHDA-induced dopaminergic degeneration was completely rescued by co-injection of an intracellular H 2 O 2 scavenger (HYDROP) or a dopamine reuptake inhibitor (GBR), suggesting that 6-OHDAmediated H 2 O 2 production in the intracellular compartment is much more critical for nigral neurodegeneration than in the extracellular compartment. 6-OHDA has been shown to produce endogenously in patients suffering from PD [30,31]. In addition to 6-OHDA, therefore, it is estimated that dopamine-mediated H 2 O 2 production in the intracellular compartment may be a key trigger for PD pathogenesis [32, 33].
In conclusion, the present study indicates that H 2 O 2 is produced by 6-OHDA taken up through dopamine transporters in the SNpc, is retrogradely transported to presynaptic glutamatergic terminals, activates TRPM2 channels, accumulates glutamate in the extracellular compartment, and rapidly induces intracellular Zn 2+ dysregulation via AMPA receptor activation, resulting in nigral dopaminergic degeneration (Fig. 8). It is likely that intracellular H 2 O 2 , but not extracellular H 2 O 2 , is a key trigger in nigral dopaminergic degeneration via intracellular Zn 2+ dysregulation and that intracellular Zn 2+ dysregulation induced by dopamine dynamics is involved in PD pathogenesis.

Declarations
Funding statement: The authors received no funding in the present paper. The present paper contains non-nancial interests. Consent to participate: The present paper has been approved by all named authors.