Norepinephrine-Fe(III)-ATP Tenary Complex and Its Relevance to

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Norepinephrine-Fe(III)-ATP Tenary Complex and Its Relevance to Parkinson’s Disease Lu Kou, Yuemei Duan, Pengcheng Wang, Yaru Fu, Narek Darabedian, Yonghui He, Dianlu Jiang, Dinglong Chen, Juan Xiang, Guokun Liu, and Feimeng Zhou ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/ acschemneuro.9b00009 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019

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ACS Chemical Neuroscience

NorepinephrineFe(III)ATP Ternary Complex and Its Relevance to Parkinson’s Disease

Lu Kou,1,

#

Yuemei Duan,1,

#

Pengcheng Wang,2 Yaru Fu,2 Nerek Darabedian,3

Yonghui He,4 Dianlu Jiang,5 Dinglong Chen,1 Juan Xiang,1, * Guokun Liu,6, * and Feimeng Zhou2, 5,*

1

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, P. R. China

2

Institute of Surface Analysis and Chemical Biology, University of Jinan, Jinan 250022, P. R. China

3

Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744 U.S.A

4

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry

of Education, Yunnan Minzu University, Kunming 650031, P. R. China 5 Department

of Chemistry and Biochemistry, California State University, Los Angeles, California

90032 U.S.A

6 State

Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen

University, Xiamen 361102, China

Corresponding Author [email protected]; [email protected]; [email protected]

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Abstract The aberrant autoxidation of norepinephrine (NE) in the presence of oxygen, which is accelerated by Fe(III), has been linked to the pathogenesis of the Parkinson’s disease (PD). Adenosine triphosphate (ATP), as a neurotransmitter whose release can be stimulated by tissue damage and oxidative stress, is co-stored and often co-released with NE in presynaptic terminals. We have shown previously that ATP inhibits the iron-catalyzed dopamine oxidation,

thereby

decreasing

the

production

of

certain

neurotoxins

such

as

6-hydroxydopamine. Whether ATP plays a similar role in Fe(III)-catalyzed NE oxidation and how it maintains the NE stability have not been investigated. Here, we studied the coordination in a ternary complex among NE, Fe(III) and ATP, and found that Fe(III) is coordinated as a octahedral center by NE and ATP. Voltammetry and mass spectrometry were employed to examine this ternary complex’s modulation of the NE autoxidation. NE−Fe(III)−ATP plays a protective role to modulate the autoxidation and Fe(III)-catelyzed oxidation of NE. The ternary complex can be detected in the substantia nigra (SN), locus coeruleus (LC), and striatum regions of C57BL/6 wild-type mice. In contrast, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse brains displayed a significant decrease of the ternary complex in the SN region and an increase in the LC and striatum areas. We posit that the ternary complex is produced by noradrenergic neurons as a protective regulator against neuronal damage and oxidative stress, contributing to the lower vulnerability of LC neurons with respect to that of SN neurons. Keywords: Norepinephrine, Adenosine triphosphate, Iron, Parkinson’s disease,


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Introduction Norepinephrine (NE) is a major catecholamine neurotransmitter in brain areas connected to physical and mental arousals and mood changes.1 Similar to dopamine, excess NE released by pre-synaptic neurons but not sequestered by post-synaptic neurons can exert toxic effects on neurons.2 The normal catabolism of NE proceeds through enzymatic pathways.3 It has been shown that, under oxidative stress, aberrant oxidation of NE to neurotoxic products (e.g., o-quinone intermediates and even neuromelanin), is accelerated by Fe(III).4 Given that iron accumulation is positively correlated with aging, neurotoxic products given rise by NE oxidation have been implicated as potential etiopathogentic species in Parkinson’s disease (PD).5,

6

Studies have demonstrated that the loss of NE in

several key brain regions ranging from locus coeruleus (LC) to cortex and hippocampus7 decreases arousal and alertness, inhibits formation and retrieval of memory, and accelerates the PD progression.8 Therefore, studies of factors that contribute to the loss of NE and the conversion of NE to neurotoxins are important to a better understanding of the etiology of PD and development of possible therapeutic treatments. Iron accumulation increases with aging.5 In PD brain, excess redox-active Fe(III) species induces aberrant oxidation of NE.9 Strong evidence has linked changes in NE and iron concentrations to the etiology of PD.10, 11 For example, semi-quantitative histochemical methods have showed that iron deposits in the neurons and glia cells of substantia nigra (SN).12 The increased level of iron is associated with neuromelanin, the end product of oxidation reactions of NE and dopamine13 and the origin of the dark blue color of LC and SN. Cell culture experiments confirmed iron-induced catecholamine neurotoxicity, in which 3 ACS Paragon Plus Environment


the coexistence of NE and iron dramatically decreases the cell viability.14 The iron-catalyzed oxidation of NE begins with the formation of the Fe(III)−NE complex in which the partial internal electron transfer from NE to Fe(III) causes the Fe(III) center to behave like Fe(II) bound to the semiquinone radicals.15 Given that the binary complex binds to O2 is necessary for the Fe(III)-catalyzed NE oxidation,16 any ligand that can bind to the NE−Fe(III) complex may at least partially block the access of O2 to the Fe(III) center and retard the Fe(III)-catalyzed NE oxidation. In cellular milieu, small ligands such as phosphate, adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) are potential candidates.17 ATP, as the main cellular currency of energy and a neurotransmitter, is abundant in many cells (>1 mM).18 Its relevance to PD has been confirmed by the substantial depletion of ATP in neuronal cells of PD brains.19 ATP has been firmly established as a co-transmitter in cell signaling.20 In dopaminergic nerve cells, dopamine (DA) is co-packaged in vesicles with ATP. Our previous work has shown that ATP inhibits the iron-catalyzed DA oxidation, thereby decreasing the production of certain neurotoxins (e.g., 6-hydroxydopamine).21 While our previous work identified the existence of the DAFe(III)ATP complex in the stratum of wide-type mouse brains, it remains unknown whether such a complex can be found in PD-model animal brains. In adrenergic nerve cells, NE is also co-packaged in vesicles with ATP. Both NE and ATP are released simultaneously during sympathetic neurotransmission.22, 23 Whether ATP plays a similar role in Fe(III)-catalyzed NE oxidation and how it modulates the NE stability have not been investigated. Therefore, the involvement of ATP in the Fe(III)-catalyzed NE oxidation reaction and its effect on the regulation of NE content in brain need to be elucidated. Of 4 ACS Paragon Plus Environment

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interest is how such a complex varies in key brain areas that undergo degenerative changes associated with Parkinsonism. Here, we investigated the iron coordination in a ternary complex formed by NE, Fe(III), and ATP using spectroscopic techniques and density functional theory (DFT) calculations. The modulation of the NE−Fe(III)−ATP complex on the autoxidation of NE was studied by voltammetric techniques. Our results showed that NE and ATP coordinate the octahedral Fe(III) center as ligands, and the resultant NE−Fe(III)−ATP ternary complex plays a protective role to modulate the autoxidation and Fe(III)-catalyzed oxidation of NE. Furthermore, we separated, identified, and quantified this complex in the SN, LC, and striatum regions of mouse brains of C57BL/6 wild-type and PD-model (induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP). The PD mice displayed significant decreases in the ternary complex content in the SN region but increases in the LC and striatum regions. Based on the biological functions of ATP, DA, and NE, as well as on some neurochemical and neuropathological findings related to PD, we posit that the NE−Fe(III)−ATP complex is produced in the exocytosis of noradrenergic neurons as a protective measure against oxidative stress and other neuropathological effects. The presence of the higher NE−Fe(III)−ATP amounts in the LC tissue likely contributes to the lower vulnerability of LC neurons with respect to that of SN neurons.

Results and Discussion Formation and Characterization of the NE−Fe(III)−ATP Complex



1.2

(NE)2-Fe(III)

0.9 Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.6

NE-Fe(III)

0.3 NE-Fe(III)-ATP

0.0 400

500

600 700 Wavelength (nm)

800

Figure 1. UV−vis spectra of NE−Fe(III) (black), (NE)2−Fe(III) (red), and NE−Fe(III)−ATP (blue) in 50 mM HEPES (pH 7.4). The Fe(III) concentration was 250 μM in the three complexes. Given that the absorption peak in catechol−metal complex is sensitive to the additional ligand coordinated to the metal center,24 we studied the formation of the NE−Fe(III)−ATP complex with absorption spectra of NE/Fe(III) mixtures with and without ATP. As shown in Figure 1, with an [NE]/[Fe(III)] ratio of 1:1, the NE−Fe(III) complex displays a characteristic absorption peak at ~560 nm corresponding to the ligand-to-metal charge transfer (LMCT) transition.25 Increasing the [NE]/[Fe(III)] ratio to 2:1, (NE)2−Fe(III) becomes the dominant species.26 It causes an enhanced characteristic absorption at ~560 nm. However,

with

the

addition

of

ATP

into

the

NE/Fe(III)

mixture

with

an

[NE]/[Fe(III)]/[ATP] ratio of 1:1:1, the characteristic absorption peak shifts to ~600 nm with an intensity comparable to that of the NE−Fe(III) complex. Since both [NE]/[ATP] and [ATP]/[Fe(III)] mixtures at an ratio of 1:1 exhibit no absorption peak in the visible region (cf. Figure S1 in Supporting Information), the shift in the absorption peak is attributed to the formation of a new ATP-containing ternary complex, while the peak intensity indicates only one NE coordinated to Fe(III) in the complex. Increasing [ATP] did not change the 6 ACS Paragon Plus Environment

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absorption peak, indicating that ATP can only replace one NE molecule in the (NE)2−Fe(III), in an analogous fashion to the DA−Fe(III)−ATP complex.21 We note that this trend is in accordance with the lower formation constant of ATP−Fe (logKf = 6.5)27 than that of NE−Fe (logKf = 7.5)28. Excess metal cations (Cu2+ and Mg2+) have no effect on the formation of the ternary complex (data not shown), which confirms the specific formation of the ternary complex.

100 +

80

+ 2+

[NE+Fe(III)+ATP-2H +Na ]

+ +

[Fe(III)+ATP-2H ]

711.9769

20

727.9935

80

732.9966 734 m/z

736

738

+

[NE+Fe(III)+ATP-2H -H2O]

20 0

400

500

600

700

800

711.9789

40

+

376.2589

+

900

+

[NE+Fe(III)+ATP-2H -H2O] 100

100

60

0

60

B

729.9882

80

728 730 732 751.9693 + + + [NE+Fe(III)+ATP-3H +Na ]

561.9214

40

Relative Abandance

729.9876

Relative Abandance

100

+ +

[NE+Fe(III)+ATP-2H ]

Relative Abandance

A Relative Abandance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60

561.9232

80 60 40 560.9132

20

559.9265

0

40

560

563.8944 563.9257

562

567.9417 568.6020

566.5858

564 566 m/z

568

570

+ +

[Fe(III)+ATP-2H ] 561.9232

20 0

1000

0

m/z

700

m/z

1400

2100

Figure 2. (A) A high-resolution mass spectrum of 10 µM NE−Fe(III)−ATP ternary complex and (B) the fragmentation spectrum of mother ions in m/z range of 729 ± 5, with m/z 729 corresponding to the molecular ion [(NE−Fe(III)−ATP)  2H+]+.

The ternary complex in a solution containing [NE]/[Fe(III)]/[ATP] of 1:1:1 was also confirmed by high-resolution mass spectrometry. As shown in Figure 2A, the molecular ion at m/z 729.9876 is in excellent agreement with that of [(NE−Fe(III)−ATP)  2H+]+ (729.9889), with an error of only 1.8 ppm. Other major peaks include Na+ adducts of the ternary

complex,

[(NE−Fe(III)−ATP)



3H+

+

Na+]+

at

m/z

751.9693

and

[(NE−Fe(III)−ATP)  2H+ + Na+]2+ at m/z 376.2589, the complex with a loss of one water molecule [(NE−Fe(III)−ATP)  2H+  H2O]+ at m/z 711.9769, and the complex with the loss 7 ACS Paragon Plus Environment


of NE, [(Fe(III)−ATP)  2H+]+ at m/z 561.9217. We also performed an MS/MS study through collision-induced dissociation with the instrument tuned to select the molecular ion at m/z 729 (Figure 2B). The two daughter ions are [NE−Fe(III)−ATP  2H+  H2O]+ at m/z 711.9769 and [(Fe(III)−ATP)  2H+]+ at m/z 561.9232. The isotopic peaks of the [(Fe(III)−ATP)  2H+]+ daughter ion (cf. inset of Figure 2B) are consistent with the presence of iron. For example, the intensity of the observed m/z of the (M2)+ ion in the inset (599.9265), which corresponds to [(Fe(III)−ATP)  2H+]+ containing the second most abundant isotope of Fe (54Fe), differs from that of the calculated m/z of 599.9270 by 0.83 ppm. All these observations strongly confirm the formation of the NE−Fe(III)−ATP ternary complex.

Structure of the NE−Fe(III)−ATP Complex A

B

0.6

C Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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D

0.4

0.2

0.0

500

600

700

800

Wavelength (nm)

Figure 3. Original model structures (A) and corresponding DFT-optimized molecular geometries (B–C) of the NE−Fe(III)−ATP complex. The purple sphere is Fe(III), yellow spheres are P, red spheres are O, blue spheres are N, gray spheres are C, and white spheres 8 ACS Paragon Plus Environment

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are H. (D) An overlay of the simulated (black curve) and experimentally measured (red curve) absorption spectra of the NE−Fe(III)−ATP complex. To gain a better understanding of the structure of the NE−Fe(III)−ATP complex, density function theory (DFT) calculation was performed. According to reports by Granot and co-worker,29, 30 possible candidates for coordinating the octahedral Fe(III) center include the three O atoms from the α-, β-, and γ-phosphate groups of ATP, two O atoms from the hydroxyl group on NE, and one O atom from H2O. Based on this, the original model structure of the ternary complex was designed (Figure 3A), and the corresponding DFT-optimized molecular geometry and the inner Fe(III) coordination sphere of the ternary complex were computed. The results are displayed in Figures 3B and 3C, respectively. The average length of the Fe–O bonds is 1.956 Å (cf. Table S1 in Supporting Information), which is close to the reported value, 1.85 Å.31 The electronic absorption spectrum (black curve), simulated by time-dependent DFT using the B3LYP functional, is overlaid with the experimental spectrum (red curve) in Figure 3D. The excellent agreements between these two curves and between the corresponding oscillator strengths indicate that the optimized molecular geometry is reasonable. A detailed analysis of the vertical excitation energies, oscillator strengths, and molecular orbital contributions (cf. Table S2 in Supporting Information) indicated that four transitions contribute to the absorption peak at 600 nm. These transitions are HOMO-2→LUMO, HOMO-2→LUMO+1, HOMO-5→LUMO+1, and HOMO-5 → LUMO. From the frontier molecular orbitals (cf. Figure S3 in Supporting Information), LUMO and LUMO+1 (the final states of the transition) are mainly located at the Fe–O bonds, while HOMO-2 and HOMO-5 (the initial states of the transition) are 9 ACS Paragon Plus Environment


largely localized at the oxygen atoms. Therefore, the electronic absorption band at 600 nm can be attributed to the O→Fe(III) LMCT.

NE−Fe(III)−ATP Complex Impedes the Catalytic Oxidization of NE by Fe(III).

Scheme 1. Proposed mechanism of the NE oxidation.

c

b

A c'

b' b

NE—Fe(III)—ATP a

c

c' b' b

1.0

a

(NE)2—Fe(III) a

NE

3.0 A

b'

-0.9

-0.6

-0.3

0.0

0.3

Potential (V)

0.6

0.9

[Leuco-NECHR]/[NE]i

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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B

(NE)2─Fe(III)

0.8 0.6

NE─Fe(III)─ATP

0.4

NE

0.2 0.0 0

20

40

60

80

100

Incubated Time (min)

Figure 4. (A) Cyclic voltammograms of NE (black), (NE)2−Fe(III) (red), and NE−Fe(III)−ATP (blue) collected from a nitrogen-purged glovebox. Scan rate = 50 mV/s. (B) Time-dependent [leuco-NECHR]/[NE]i in a O2-saturated solution of 250 μM NE (■), and in the presence of 125 μM Fe(III) (▲) or 250 μM Fe(III) and 250 µM ATP (●). To elucidate the effect of ATP on the iron-catalyzed oxidation of NE, we studied the electrochemical behaviors of NE, (NE)2−Fe(III), and NE−Fe(III)−ATP. As shown in Figure 4A, in the absence of Fe(III), NE is oxidized to NE-o-quinone at ~0.24 V (peak a), which 10 ACS Paragon Plus Environment

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rapidly cyclizes to leuconoradrenochrome (leuco-NECHR) as depicted in Scheme 1. This was then followed by further oxidation to neuromelanin and noradrenolutin via two different intermediates, yielding peaks b and b’, respectively.32 Neuromelanin as an end product is the origin of the blue or dark cell color in the LC region.1 In the mixture of NE and Fe(III), the potentials of peaks b and b’ did not change. However, two shoulder peaks (peaks c and c’) emerged, which can be ascribed to the redox reaction of Fe(III)/Fe(II).21 The NE oxidation in the (NE)2−Fe(III) complex appears at 0.38 V, which is 0.16 V more positive than the NE oxidation peak. Such a shift indicates that NE is more difficult to oxidize when bound to Fe(III), owing to the partial distribution of the NE electron density to Fe(III). Concurrently, the reduction peak of Fe(III) appears at −0.38 V, similar to our earlier report on the DA−Fe(III)−ATP complex.21 Finally, the additional ligation of ATP in the NE−Fe(III)−ATP complex causes the positive shift of the NE oxidation peak potential to 0.47 V, and the negative shift of the Fe(III) reduction to −0.42 V, respectively. Thus, ATP attenuates the reducing and oxidizing power of NE and Fe(III) in the NE−Fe(III)−ATP, rendering NE more difficult to oxidize and Fe(III) more difficult to reduce. We also evaluated the effect of ATP on the iron-catalyzed NE oxidation by oxygen. To monitor NE consumption, we quantified the intermediate leuco-NECHR, which is the precursor of neuromelanin, by using the area of the reduction peak (peaks b’) obtained with differential pulse voltammetry (DPV) in oxygen-saturated solutions of NE, (NE)2−Fe(III), or NE−Fe(III)−ATP. Figure 4B displays the [leuco-NECHR]time curves collected from DPV scans (cf. Figure S4 in Supporting Information). Note that the leuco-NECHR concentrations are normalized with respect to the initial [NE] ([NE]i). The three plateaus correspond to 11 ACS Paragon Plus Environment


46%, 65% and 90% of [NE]i. The total amount of leuco-NECHR produced in the NE−Fe(III)−ATP solution is 25% less than that in the (NE)2−Fe(III) solution. Thus, Fe(III) catalyzes the NE oxidation by O2 and the ATP ligation significantly inhibits the Fe(III)-catalyzed NE oxidation. In our previous work, we found that ATP exhibits a similar coordination chemistry towards the (DA)2−Fe(III) complex.21 In doing so, binding of O2 to the Fe(III) center is prevented and the DA oxidation is retarded.21 In addition, production of neurotoxic DA oxidation intermediates such as 6-OHDA is curtailed. Given the highly analogous structures between DA and NE, it appears that the same effect is at work for the NE−Fe(III)−ATP complex. The partial internal electron transfer from the ligands causes the Fe(III) center to behave as Fe(II) bound to the semiquinone radicals.15 Molecular O2 can easily access and bind to the (NE)2−Fe(III) by replacing two weakly bound H2O molecules (bond length 2.044 Å) at the distal sites,21 and subsequently accelerates the iron-catalyzed NE oxidation.16 Consequently, the (NE)2−Fe(III)−O2 dissociates into hydrogen peroxide, Fe(III), and NE-o-quinone, which rapidly cyclizes to leuco-NECHR. ATP, as a tetradentate ligand, can replace one NE molecule and one weakly bound H2O molecule when binding to (NE)2−Fe(III). The octahedral Fe(III) coordinates with NE and ATP to form a rather stable ternary complex with the two hydroxyl oxygen atoms of NE and α, β, γ-phosphate oxygen atoms of ATP. Even though the sixth ligand is the weakly bound H2O molecule at the distal site (bond length 1.998 Å), the compact structure is less susceptible to O2 binding. In addition, from our computational results, ATP in the NE−Fe(III)−ATP complex redistributes the electron density more effectively than the second NE ligand in the (NE)2−Fe(III) 12 ACS Paragon Plus Environment

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complex, affording the former a lower accessibility of the Fe(III) center by O2. In short, the coordination chemistry and our computational results confirm that the formation of NE−Fe(III)−ATP complex retards NE autoxidation in the presence of O2.

Identification and Quantitation of the NEFe(III)ATP Complex in Brain

A

PD Relative Abandance

Relative Abandance

Tissues of Wild-type and PD-model Mice.

+

748 [M]

740

744

748

m/z

752

756

C 25

1.5 1.2 ATP (nmol/g)

15 10

PD 374 [M]2+

370

WT PD

20

B

760

30 NE-Fe(III)-ATP (pmol/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


372

374

m/z

376

D

378

380

WT PD

0.9 0.6 0.3

5 0

0.0

SN

Striatum

LC

SN

Striatum

LC

Figure 5. Mass spectra of tissues extracted from the SN region of an MPTP-induced PD mouse brain showing (A) the singly charged and (B) doubly charged NE−Fe(III)−ATP complex ions. (C) The NE−Fe(III)−ATP levels in the SN, striatum, and LC regions of the C57BL/6 wild-type and PD-model mice. The ternary complex was quantified by the peak at m/z 374 and normalized with respect to the signal of an internal standard nimodipine (100 ng/mL). Error bars represent the standard deviations of three parallel experiments. (D) The ATP levels in the SN, striatum, and LC regions of C57BL/6 wild-type and PD-model mice. The data represent the means and standard deviations of three parallel experiments. Given the high abundances of ATP and iron in the brain and the stability of the NE−Fe(III)−ATP ternary complex, we successfully separated, identified, and quantified this 13 ACS Paragon Plus Environment


complex in tissues extracted from the NE-rich regions of mouse brains. We selected the LC, striatum, and SN regions based on the considerations that (1) NE is most abundant in LC, which is one of the brainstem structures that undergo degenerative changes in early PD,33 (2) prior to clinical Parkinsonism, the striatal motor area experiences loss of dopaminergic cells, and (3) the prominent motor abnormality in late PD is related to neuronal cell loss in SN and the subsequent loss of dopamine in basal ganglia.34 Figure 5A displays a representative mass spectrum of tissues extracted from the SN region of an MPTP-induced PD mouse brain. The peak at m/z 748 is in good agreement with the theoretical molecular weight of the [(NE−Fe(III)−ATP)  2H+ + H2O]+ (748.2255). Additionally, the peak at m/z 374 (Figure 5B) is likely the doubly charged ion created either in the electrospray or in the gas phase. We should note that the water adduct of this ternary complex was observed with our triple-quadrupole mass spectrometer, instead of the [(NE−Fe(III)−ATP)  2H+]+ ion revealed by the high-resolution mass spectrometer (cf. Figure 2). The formation of a water adduct in the former is probably due to the differences in ionizations and the different instrumental designs (triple quadrupole MS vs. the Orbitrap-based high-resolution MS). The assignment of these MS peaks to NE−Fe(III)−ATP was further confirmed by the increases of these two peaks obtained after performing liquid chromatography-MS (LC-MS) experiment of an tissue extract spiked with a standard ternary complex (cf. Figure S5 in Supporting Information). The ternary complex was readily detectable in the SN, LC, and stratum tissues extracted from the wild-type mouse brain (blue bars in Figure 5C). The NE−Fe(III)−ATP ternary complex in different brain tissues was quantified using the internal standard method 14 ACS Paragon Plus Environment

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by integrating the mass spectra peak and normalizing the intensity with respect to that of the internal standard nimodipine (100 ng/mL).35 Notice that the NE−Fe(III)−ATP levels between the wild-type and the PD-model mouse brains are markedly different in the three brain regions. Specifically, the SN region in the wild-type mouse brains exhibited a ternary complex concentration that is 166.8% greater than that in the PD-model mouse brains. In contrast, the ternary complex in the striatum and LC regions of the wild-type mice is 24.0% and 21.0% as high as that of the PD model mice, respectively. As ATP contributes to the formation of the ternary complex, we also determined the total ATP level in these three brain regions. We first attempted to use LC-MS but only to find that ATP, being highly polar, was difficult to detect as it elutes out of the column too fast. We therefore resorted to biomiluminescence measurements using a plate reader. From the data in Figure 5D, it is apparent that (1) the ATP levels in the three brain regions of the PD mice are all considerably lower than the respective counterparts of the wild-type mice, and (2) while the ATP levels in wild-type mouse brains vary between the three regions, the levels in the PD mouse brains, do not vary as significantly. The results presented in Figures 4 and 5, when put in the context of our earlier findings on the biological functions and implication of the DA−Fe(III)−ATP complex, help form a more comprehensive description about the role of ATP in maintaining the catecholamine neurotransmitter concentrations and regulating the oxidative stress inherent in aberrant chemical oxidation of these neurotransmitters. The first similarity between the DA−Fe(III)−ATP and NE−Fe(III)−ATP complexes is that, ATP not only curtails the unwanted DA or NE degradation, but also effectively prevents neurotoxic intermediates 15 ACS Paragon Plus Environment


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from forming. For the former case, the intermediate 6-OHDA has been firmly established as a neurotoxin.36 For the latter, strong evidence has indicated that noradrenochrome, as one of the intermediates in Scheme 1, is also a potent neurotoxin because it can readily conjugate with thiol- and amino-containing amino acids, with the adducts eventually being accumulated into neuromelanin.37 Furthermore, although the biological function of neuromelanin is controversial and conflicting results on its neuroprotective and neurotoxic behaviors have been reported, it is becoming evident that over-accumulation of toxic species such as iron and adducts of o-quinones can be detrimental.37,

38

Catecholamine

neurotransmitters can be neurotoxic when present in high concentrations, and cells have developed different mechanisms to metabolize excess neurotransmitters that are not sequestered by postsynaptic neuronal receptors or recaptured by presynaptic neuronal receptors

(e.g.,

through

enzymatic

digestions

by

monoamine

oxidase39

and

catechol-O-methyltransferase40 or up-taken by glial cells41). The fact that the NE−Fe(III)−ATP complex is present in areas where NE exists suggests that formation of this complex could be via a non-enzymatic route to limit accumulation of toxic intermediates and products in neuromelanin. The second resemblance between the DA−Fe(III)−ATP and NE−Fe(III)−ATP complexes is their formations. In vesicles, both are co-present in a complex with ATP and acidic proteins known as chromagranins.20,

42

Moreover, it has been reported that iron is complexed by at least some NE molecules and DA in vesicles.21, 43 It is well known that iron loading increases with aging and the problem is particularly acute in neurological disorders.44 When these neurotransmitters are released with ATP from the vesicles after the arrival of an action potential, without formation of the 16 ACS Paragon Plus Environment

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DA−Fe(III)−ATP and NE−Fe(III)−ATP complexes, the iron-catalyzed catechol oxidation could produce undesirable neurotoxins between neurons. Perhaps the most interesting and yet seemingly paradoxical finding of our work is the markedly different distributions of the ternary complexes between the wild-type and PD-model mouse brains in the three regions examined (Figure 5C). The trend observed in the

SN

is

expected,

as

the

extremely

toxic

metabolite

of

MPTP,

1-methyl-4-phenylpyridinium (MPP+), can be easily transported by the dopamine uptake system into dopaminergic neurons,45, 46 and treatment of mice disrupts the electron transport chain and severely impedes the ATP formation47. Inhibition of tyrosine hydroxylase was also found to reduce DA in SN cells significantly.48 As DA is a precursor of NE, a significant reduction of NE will also decrease the amount of NE−Fe(III)−ATP. Thus, even though the iron bioavailability to SN is greater than those in the LC and stratum regions,44, 49 the overall NE−Fe(III)−ATP concentration is lower in the SN region of the PD-model mice due to the decreased NE concentration. Noradrenergic neurons are most abundant in the LC region and MPP+ also has high affinity toward norepinephrine uptake sites. If extensive damages had occurred, NE and ATP levels in LC and striatum regions would decline significantly. As a result, the NE−Fe(III)−ATP complex would be of lower concentrations in these regions of the PD-model mice than the counterparts of the wild-type. However, minimal damages to noradrenergic neurons were reported.50 Similarly, a striking recovery of striatal dopamine levels has been shown after MPTP administrations.51 Therefore, the NE levels in the LC and striatal regions do not vary under the assault of MPP+. We rationalize that the increases of the ternary complex in the LC and striatal regions of PD mice are due to 17 ACS Paragon Plus Environment


the increased release of ATP from the vesicles to cytosol. This is because that ATP release can be triggered by a wide range of cell types to activate receptors on sensory afferents to initiate pain that results from tissue damage, oxidative stress, and nerve injury.34 We should add that this rationale is not at odds with the declined ATP levels shown in Figure 5D. As reviewed by Erecińska and Silver,52 ATP is mainly produced in mitochondria via oxidative phosphorylation of adenosine diphosphate. Upon entering cells, MPP+ accumulates in the mitochondria and impairs the ATP production.46 This is why in the three brain regions of the PD mice, ATP levels all decreased (Figure 5D). In contrast, when MPP+ is accumulated into synaptosomal vesicles, cells can become MPP+-resistant.53 Thus, ATP packaged with NE will not be decreased upon the entry of MPP+ into vesicles. Lack of the Fe(III) coordination with ATP, however, will accelerate the iron-catalyzed NE oxidation when iron overloading occurs. That ATP is co-stored and co-released as a neurotransmitter with NE in LC neurons42 lends additional support to this contention. We estimated that the ternary complex in the three brain regions of PD mice ranges between 8.2 ± 1.3 and 15.3 ± 3.2 pmol/g tissue, which are about 1.4 ± 0.3% in SN, 1.7 ± 0.4% in striatum and 3.0 ± 0.7% in LC of the total ATP. Although we cannot completely rule out that some ternary complexes were formed during the cell homogenization step, we believe that this is an unlikely event as in cellular milieu the free iron concentration is exceedingly low (

Norepinephrine-Fe(III)-ATP Tenary Complex and Its Relevance to

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