Post-treatment with an ultra-low dose ofNADPH oxidase inhibitor diphenyleneiodoniumattenuates disease progression in multipleParkinsons disease models

Qingshan Wang,1 Li Qian,1 Shih-Heng Chen,1 Chun-Hsien Chu,1 Belinda Wilson,1

Esteban Oyarzabal,1 Syed Ali,2 Bonnie Robinson,2 Deepa Rao3 and Jau-Shyong Hong1

Nicotinamide adenine dinucleotide phosphate oxidase, a key superoxide-producing enzyme, plays a critical role in microglia-

mediated chronic neuroinammation and subsequent progressive dopaminergic neurodegeneration in Parkinsons disease.

Although nicotinamide adenine dinucleotide phosphate oxidase-targeting anti-inammatory therapy for Parkinsons disease has

been proposed, its application in translational research remains limited. The aim of this study was to obtain preclinical evidence

supporting this therapeutic strategy by testing the efcacy of an ultra-low dose of the nicotinamide adenine dinucleotide phosphate

oxidase inhibitor diphenyleneiodonium in both endotoxin (lipopolysaccharide)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-

treated mice using post-treatment regimens. Our data revealed that post-treatment with diphenyleneiodonium signicantly

attenuated progressive dopaminergic degeneration and improved rotarod activity. Remarkably, post-treatment with diphenyleneio-

donium 10 months after lipopolysaccharide injection when mice had 30% loss of nigral dopaminergic neurons, showed high

efcacy in protecting the remaining neuronal population and restoring motor function. Diphenyleneiodonium-elicited neuroprotec-

tion was associated with the inhibition of microglial activation, a reduction in the expression of proinammatory factors and an

attenuation of a-synuclein aggregation. A pathophysiological evaluation of diphenyleneiodonium-treated mice, including assess-

ment of body weight, organs health, and neuronal counts, revealed no overt signs of toxicity. In summary, infusion of ultra-low

dose diphenyleneiodonium potently reduced microglia-mediated chronic neuroinammation by selectively inhibiting nicotinamide

adenine dinucleotide phosphate oxidase and halted the progression of neurodegeneration in mouse models of Parkinsons disease.

The robust neuroprotective effects and lack of apparent toxic side effects suggest that diphenyleneiodonium at ultra-low dose may

be a promising candidate for future clinical trials in Parkinsons disease patients.

1 Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, ResearchTriangle Park, NC 27709, USA

2 Neurochemistry Laboratory, Division of Neurotoxicology, National Centre for Toxicological Research/USFDA, Jefferson, AR72079, USA

3 National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina27709, USA

Correspondence to: Qingshan Wang,

National Institute of Environmental Health Sciences,

111 T.W. Alexander Dr.,

Research Triangle Park,

North Carolina, 27709, USA

E-mail: wangq4@niehs.nih.gov

Correspondence may also be addressed to: Jau-Shyong Hong. E-mail: hong3@niehs.nih.gov.

doi:10.1093/brain/awv034 BRAIN 2015: 138; 12471262 | 1247

Received September 12, 2014. Revised November 25, 2014. Accepted December 16, 2014. Advance Access publication February 25, 2015

Published by Oxford University Press on behalf of the Guarantors of Brain 2015. This work is written by US Government employees and is in the public domain in the US

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Keywords: Parkinsons disease; microglia; NADPH oxidase; neuroinammation; superoxide

Abbreviations: DOPAC = dihydroxyphenylacetic acid; DPI = diphenyleneiodonium; LPS = lipopolysaccharide;MPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NADPH = nicotinamide adenine dinucleotide phosphate

IntroductionParkinsons disease is an age-associated movement disorder

that progresses over decades in aficted individuals.

The pathological hallmark of Parkinsons disease is the

progressive nigrostriatal dopaminergic neurodegeneration

coupled with intracellular inclusions known as Lewy

bodies (Olanow and Tatton, 1999). Current clinical dopa-

mine replacement interventions for patients with

Parkinsons disease provide temporary symptomatic relief

but fail to halt disease progression (Salawu et al., 2010).

Thus, alternative strategies must be developed to target

Parkinsons disease progression to modify the course of

the disease.

Microglia-mediated neuroinammation has been linked

to multiple neurodegenerative diseases, including

Parkinsons disease (Gao et al., 2002, 2003; Gao and

Hong, 2008; Perry et al., 2010; Czirr and Wyss-Coray,

2012; Phani et al., 2012). These ndings prompted

pharmaceutical companies to investigate the use of anti-

inammatory drugs as potential treatments for

Parkinsons disease. Early epidemiological and animal stu-

dies supported that non-steroidal anti-inammatory drugs

have been shown to reduce the risk of acquiring

Parkinsons disease (Teismann and Ferger, 2001; Chen

et al., 2005). However, recent meta-analyses and

case-control studies failed to support these ndings (Samii

et al., 2009; Becker et al., 2011). The development of novel

anti-inammatory strategies to treat neurodegenerative dis-

eases has been further hampered by the failure of several

clinical trials (McGeer and McGeer, 2007). The inability of

translating successful strategies from animal studies to

human therapy highlighted the need for better therapeutic

strategies and more suitable animal models in Parkinsons

disease therapy development.

One recent strategy for Parkinsons disease therapy has

been to deviate from conventional anti-inammatory tar-

gets and inhibit upstream mediators, such as microglial

nicotinamide adenine dinucleotide phosphate (NADPH)

oxidase (Gao et al., 2012), a key superoxide-producing

enzyme. Once activated, NADPH oxidase produces extra-

cellular and intracellular reactive oxygen species (Lambeth,

2004), which are critical in initiating and maintaining

chronic neuroinammatory responses, leading to progres-

sive dopaminergic neurodegeneration (Block et al., 2007;

Gao and Hong, 2008; Lambeth et al., 2008). As a proof

of concept, we used the NADPH oxidase inhibitor diphe-

nyleneiodonium (DPI) as a therapy for Parkinsons disease.

Although DPI lacks clinical use at its recommended dose

(mg/kg) because of non-specicity and high toxicity (Aldieri

et al., 2008), we recently reported that DPI at

sub-picomolar concentrations (1014 to 1013M) specic-ally inhibits NADPH oxidase activation and protects dopa-

minergic neurons in vitro (Wang et al., 2014a). Beyond

using a new class of anti-inammatory drugs,

we recognized that the choice of suitable animal models

was essential for the successful development of

Parkinsons disease therapies. Widely used parkinsonian ani-

mal models, including those generated by

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or

6-hydroxydopamine, acutely lesion dopaminergic neurons

within days but fail to recapitulate the progressive feature

of Parkinsons disease (Cannon and Greenamyre, 2010).

Moreover, the use of pretreatment regimens for candidate

therapies in these acute parkinsonian animal models to-

gether with their toxicities after long-term usage have ham-

pered the progress of successful translational therapies.

Here, we aimed to overcome these obstacles by

post-administering an ultra-low dose of DPI in chronic in-

ammation-based Parkinsons disease models, which recap-

itulate the delayed, progressive features of dopaminergic

degeneration. We found that DPI at an extreme low dose

(10 ng/kg/day, subcutaneously for 2-week infusion) showed

no apparent toxicity and successfully protected dopamin-

ergic neurons in two inammation-driven mouse models

after the loss of 30% dopaminergic neurons. Thetherapeutic potential of DPI was further veried in a sub-

chronic MPTP Parkinsons disease mouse model. Parallel

experiments in NADPH oxidase-decient mice validated

that NADPH oxidase is the molecular target of DPI-elicited

neuroprotection. Our ndings suggest a promising strat-

egy for arresting Parkinsons disease progression by miti-

gating neuroinammation through the inhibition of

NADPH oxidase.

Materials and methods

Animal treatments

A repeated MPTP regimen (15mg/kg, subcutaneously for sixconsecutive days) or a single systemic lipopolysaccharide (LPS)injection (Escherichia coli 0111:B4, Sigma) were administeredto C57BL/6J and/or transgenic mice over-expressing humanA53T mutant -synuclein (B6.C3-Tg [Prnp-SNCA*A53T]83Vle/J, The Jackson Laboratory) mice. The dosage ofMPTP (Zhang et al., 2004; Wang et al., 2014b) or LPS (Qinet al., 2007, 2013) was selected based on our previous studies.Mice used as vehicle controls received an equal volume of0.9% saline. In both MPTP and LPS regimens, mice weretreated subcutaneously with DPI at 10 ng/kg/day for 2 weeksvia an Alzet osmotic pump. In LPS-treated C57BL/6J mice,DPI was administered after 3 months (pre-motor group,

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n = 8 to 11 each group with total 37 mice) or 10 months(motor group, n = 6 each group with total 24 mice) afterLPS injection. LPS-treated transgenic mice over-expressinghuman A53T mutant -synuclein (n = 5 to 6 each groupwith total 16 mice) received DPI infusion after 1 month ofLPS challenge. In MPTP-injected C57BL/6J mice (n = 8 to 10each group with total 27 mice), DPI infusion started 3 daysafter the rst injection of MPTP. All the mice were euthanizedat the desired time points. Housing, breeding and experimentaluse of the animals were performed in strict accordance withthe National Institutes of Health guidelines. All procedureswere approved by the National Institute of EnvironmentalHealth Sciences/National Institutes of Health animal care anduse committee.

Immunohistochemistry and double-labelling immunofluorescence

Immunostaining and double-labelling immunouorescencewere performed as described previously (Gao et al., 2011;Wang et al., 2012, 2014b). For details of immunostainingassays and quantitative analysis see the online Supplementarymaterial.

Cell counts

The number of tyrosine hydroxylase-immunoreactive neuronsin the substantia nigra pars compacta was estimated usingstereological methodology with the optical fractionatorsmethod (MBF Science) as described previously (Wang et al.,2014b). For details see Supplementary material.

Rotarod test

The rotarod behaviour test was measured using a Rotamexdevice (Columbus Instruments). The parameters of therotarod system include start speed, acceleration and highestspeed (1 rpm, accelerate 12 rpm/2 s, 50 rpm). The miceunderwent three consecutive trials. The rest period betweeneach trial was 30min. The mean latency time to fall offthe rotating rod for the last two trials was used for theanalysis.

In situ visualization of superoxide andsuperoxide-derived oxidantproduction

In situ visualization of oxidative stress was assessed bydihydroethidium histochemistry according to previous reportswith minor modications (Quick and Dugan, 2001; Wu et al.,2003). Briey, LPS-injected mice were administered singleinjections [intraperitoneally (i.p.)] of dihydroethidium ata dose of 20mg/kg. Eighteen hours later, mice were perfusedtranscardially with PBS, and coronal substantia nigra sectionswere examined for the dihydroethidium productusing uorescence microscopy (excitation 534 nm; emission580 nm).

Real-time PCR analysis

Total RNA was extracted with the RNeasy Mini kit andreverse-transcribed with an oligo dT primer. Real-timePCR amplication was performed using SYBR Green PCRMaster Mix (Applied Biosystems) and Applied Biosystems7900HT Fast Real-Time PCR System according to the manu-facturers protocols. The primers were designed by Vector NTIVersion: Advance 11 software (Invitrogen, SupplementaryTable 1). The PCR conditions were 95C for 10 s, 55C for30 s, and 72C for 30 s for 40 cycles. All of the data werenormalized to Gadph.

Catecholamine content analysis

The levels of dopamine and its metabolite dihydroxyphenyla-cetic acid (DOPAC) were measured using high-performanceliquid chromatography and coupled with electrochemical de-tection as described previously (Zhang et al., 2004). For detailssee Supplementary material.

Statistical analysis

Data are expressed as the mean standard error of the mean(SEM) and were analysed statistically with Graph-Pad Prism(GraphPad Software Inc.). Differences with two groups wereanalysed using unpaired two-tailed Students t-test. Formore than two groups, one- or two-way ANOVA was applied.When ANOVA showed signicant differences, pair-wise com-parisons between means were tested by Tukeys post hoctesting. In all analyses, P50.05 was considered statisticallysignicant.

Results

Post-treatment with an ultra-lowdose of DPI prevents dopaminergicneurodegeneration and motordeficits in LPS-treated mice

A neuroinammation-driven progressive dopaminergic neu-

rodegenerative model generated by a single systemic injec-

tion of the endotoxin LPS (Qin et al., 2007) was used to

determine whether post-treatment of DPI can halt disease

progression. The advantages of using this inammation-

based model are twofold: (i) progressive nigral dopamin-

ergic neuron loss over a period of 10 months after LPS

injection coincides with motor decits that can be reversed

by L-DOPA (Qin et al., 2007, Liu et al., 2008); and (ii)

mimicking the therapeutic window for Parkinsons disease

patients, treatment can be performed at different stages of

disease progression. In the present study, LPS-treated mice

were separated into two treatment groups (Fig. 1A). The

rst group was treated with DPI 3 months after LPS injec-

tion, a time point prior to nigral dopaminergic neuron loss

and behavioral decits (premotor stage). The second group

was treated with DPI after 10 months of LPS injection,

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when signicant dopaminergic neurodegeneration (3035%

loss of neurons) and motor decits were observed (motor

stage). An ultra-low dose of DPI (10 ng/kg/day), which is

approximately one-millionth of the standard doses used in

previous reports (Vlessis et al., 1995; Miesel et al., 1996),

was delivered systemically via a subcutaneously implanted

mini-pump for 2 weeks. This ultra-low dose was chosen

based on exploratory dose-response studies evaluating the

effectiveness of doses from 0.1 to 10 ng/kg. We found that

10 ng/kg was the lowest dose that still showed potent neu-

roprotection. Although DPI is known to readily cross the

bloodbrain barrier (Gatley and Martin, 1979), the meas-

urement of the DPI concentration in the brain at this dose

was beyond the detection sensitivity of the analysis.

By extrapolating the pharmacokinetic data from a previous

report (Gatley and Martin, 1979), we estimated the brain

concentration of DPI to be in the subpicomolar range,

which is comparable to the in vitro dose we used previously

(Wang et al., 2014a). The efcacies of this ultra-low DPI

on LPS-induced dopaminergic neuron loss and reduction in

rotarod activity were determined 7 months after infusion

for both the premotor (i.e. total 10 months after LPS injec-

tion) and motor groups (i.e. total 17 months after LPS

injection) (Fig. 1A). This time point was selected because

dopaminergic neurodegeneration in premotor-group mice is

much more evident after 10 months of LPS injection (Qin

et al., 2007, 2013). For consistency, we also selected the

same time point (7 months after DPI infusion) in motor-

group mice.

Consistent with our previous report (Qin et al., 2007),

LPS injection reduced the number of nigral tyrosine

hydroxylase-immunoreactive neurons in premotor stage

mice by 32%. An almost 50% loss of tyrosine

hydroxylase-immunoreactive neuron was detected in

motor stage mice (Fig. 1B and C). These results replicated

our previous nding showing progressive dopaminergic

neurodegeneration in LPS-injected mice (Qin et al., 2007,

2013). It is important to note that the loss of nigral dopa-

minergic neurons was previously conrmed to reect the

death of the neurons rather than the loss of tyrosine hydro-

xylase immunoreactivity (Gao et al., 2011). DPI

post-treatment exhibited signicant protection against

LPS-induced dopaminergic neuron loss in both premotor

(P = 0.0002, compared with LPS alone group) and motor

(P = 0.0339, compared with LPS alone group) stage groups.

These results indicate that the LPS-elicited progressive

Figure 1 Post-treatment with an ultra-low dose of DPI attenuates dopaminergic neurodegeneration and motor deficits in

LPS-treated mice. (A) Experimental designs. C57BL/6J mice received a single injection of LPS [15 106 EU/kg, intraperitoneally (i.p.)]. Three(pre-motor stage) or 10 (motor stage) months after LPS injection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously)

via osmotic mini-pump for 2 weeks. Measurements of neuron loss and motor deficits were performed 7 months after DPI infusion. (B) Seven

months after DPI treatment, dopaminergic neurons in the substantia nigra pars compacta were immunostained with anti-tyrosine hydroxylase

antibody and representative images are shown. (C) The number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars

compacta was counted stereologically. (D) The effects of ultra-low-dose DPI on LPS-induced motor deficits were measured using the rotarod

test. Data are expressed as the mean SEM and were analysed by two-way ANOVA followed by Tukeys post hoc testing. *P5 0.05, **P5 0.01;#P5 0.05, ##P5 0.01; n = 611; Scale bar = 200 mm. Con = control; TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.

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neurodegenerative process was attenuated by DPI. In con-

currence with protecting neuronal cell bodies, DPI post-

treatment also maintained the integrity of the neurite

network of dopaminergic neurons in the substantia nigra

pars reticulata, as demonstrated by greater tyrosine hydro-

xylase-immunoreactive bre density and a reduction of

dendritic beading (fragmented dendrites) in DPI/LPS-treated

mice compared with the LPS alone group (Supplementary

Fig. 1A). Consistent with these morphological observations,

the LPS-induced decrease of striatal dopamine levels was

signicantly attenuated by DPI post-treatment, which

showed no differences compared with the vehicle controls

(Supplementary Fig. 1B).

Post-treatment with an ultra-low dose of DPI not only

showed signicant neuroprotection but, even more impres-

sively, displayed potent efcacy in attenuating LPS-elicited

motor decits in both the pre-motor and motor groups.

DPI post-treatment markedly attenuated the LPS-induced

reduction of rotarod activity in both the premotor

(P = 0.0132 compared with LPS alone group) and motor

group (P = 0.0072, compared with LPS alone group)

(Fig. 1D). It is interesting to note that DPI post-treatment

was able to restore the rotarod activity of LPS-treated mice

to the same level as control mice, despite its inability to

fully prevent the loss of nigral dopaminergic neurons.

These results suggest that ultra-low dose DPI is capable

of halting progressive dopaminergic neurodegeneration

and motor decits.

Post-treatment with an ultra-lowdose of DPI is neuroprotective inLPS-treated human A53T a-synucleinover-expressing mice

-Synuclein is a major constituent of Lewy bodies in patients

with Parkinsons disease. The A53T mutation in the SNCA

gene is known to increase the amount of aggregated -synu-

clein and is closely associated with dopaminergic neurode-

generation (Lee and Trojanowski, 2006). Although

transgenic mice over-expressing human A53T mutant -

synuclein have been previously used as rodent models of

Parkinsons disease, reports have shown that this transgenic

mouse fails to develop overt nigral dopaminergic neurode-

generation (Giasson et al., 2002; Gao et al., 2011). We re-

cently developed a two-hit model by injecting LPS into

transgenic mice over-expressing human A53T mutant -

synuclein, which showed earlier onset and a much more

robust progressive loss of nigral dopaminergic neurons than

C57BL/6J mice (Gao et al., 2011). Thus, the LPS-injected

transgenic mouse can serve as an ideal model with which to

investigate the neuroprotective effects of DPI. Because of the

early onset (as early as 2.5 months after LPS injection) of

dopaminergic neurodegeneration in LPS-treated transgenic

mice over-expressing human A53T mutant -synuclein

(Gao et al., 2011), the protective effects of DPI were deter-

mined after 4 months of LPS injection, a time point that

showed 4050% loss of dopaminergic neurons. Consistent

with previous report (Gao et al., 2011), 4 months after LPS

injection, a 43% loss of nigral dopaminergic neurons was

noted in transgenic mice over-expressing human A53T

mutant -synuclein compared to untreated littermate con-

trols. Post-treatment with DPI 1 month after LPS injection

led to a signicant protection (P = 0.0029, compared with

LPS alone group) of the nigral tyrosine hydroxylase-immu-

noreactive neurons (Fig. 2AC). A similar degree of DPI-eli-

cited protection of dopaminergic terminals in the striatum

was also observed, as shown by the high striatal tyrosine

hydroxylase density in the DPI-treated group (87 4% ofcontrol) compared to the non-DPI-treated group (56 7%,P = 0.0006; Fig. 2B and D).

The expression of human -synuclein in transgenic mice

over-expressing human A53T mutant -synuclein is not

homogeneous in the brain with high levels in brainstem

and cortex and very low levels in the substantia nigra

(Giasson et al., 2002). We recently conrmed this nding

showing minimal protein expression in the substantia nigra

by western blot and immunohistochemistry using an

antibody again human -synuclein in untreated transgenic

mice over-expressing human A53T mutant -synuclein

(Gao et al., 2011). Interestingly, after LPS injection,

increased expression and insolubility of human -synuclein

were observed in the substantia nigra (Gao et al., 2011).

In addition to protecting dopaminergic neurons, DPI

post-treatment mitigated LPS-induced human -synuclein

accumulation in the substantia nigra, as detected by immu-

nostaining using SYN211 antibody (specic for human

-synuclein, Fig. 3A). Double-label immunouores-

cence analysis revealed an accumulation of -synuclein

in the cytosol and perinuclear locations of nigral tyrosine

hydroxylase-immunoreactive neurons after LPS injection. In

contrast, nigral -synuclein immunoreactivity in DPI/LPS-

treated transgenic mice over-expressing human A53T

mutant -synuclein was diffuse and barely visible

(Fig. 3B). Quantitative analysis of the nigral SYN211

density indicated a 223% increase in the LPS alone

group, which was reduced to 127% in DPI/LPS-treated

mice (P5 0.0001, compared with LPS alone group;Fig. 3C).

Post-treatment with an ultra-lowdose of DPI attenuates LPS-elicitedmicroglia-mediatedneuroinflammation

To determine whether the neuroprotective effects of DPI

were related to its anti-inammatory properties, we exam-

ined the inhibitory effects of DPI on microglial activation

using post-treatment regimens at different stages of the

neurodegenerative process. Activation of microglia in

the subsantia nigra was morphologically observed by

immunostaining with two microglial markers: ionized cal-

cium binding adaptor molecule 1 (AIF1, also known as

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Iba-1) and ITGAM (formerly known as CD11b, alpha

chain of the b-2 integrin receptor). In both the premotorand motor stage, activated microglia characterized by a

hypertrophied morphology and intensied ITGAM and

AIF1 staining were observed throughout the nigral reticu-

lata area (Fig. 4A). Analysis of ITGAM and AIF1 density

and cell body size supported these morphological observa-

tions. Compared with the LPS alone group, post-treatment

with DPI markedly attenuated microglial activation, as

shown by a reduced density of ITGAM (P = 0.0020 and

0.0001 in premotor and motor stage, respectively) and

AIF1 (P = 0.0198 and 0.0001 in premotor and motor

stage, respectively) staining and microglial cell body sizes

(P50.0001 and 0.0001 in premotor and motor stage, re-spectively; Fig. 4BD).

Activated microglia secrete a variety of toxic factors, such

as tumor necrosis factor alpha, interleukin-1 beta and other

proinammatory cytokines, which work in concert to cause

neuronal damage (Block and Hong, 2005). Exposure to

LPS produced a long-lasting 2-fold increase in the

expression of the proinammatory genes tumor necrosis

factor alpha, interleukin-1 beta and major histocompatibil-

ity complex II, in both the premotor and motor groups.

Interestingly, ultra-low dose DPI post-treatment prevented

the LPS-induced increase in the gene expression of these

immune factors (Fig. 4EG).

Post-treatment with an ultra-lowdose of DPI inhibits LPS-inducedoxidative stress

NADPH oxidase is essential for maintaining chronic micro-

glia-mediated neuroinammation and subsequent progres-

sive dopaminergic neurodegeneration (Qin et al., 2013).

Although DPI exhibits potent inhibitory effects on

NADPH oxidase at mg/kg doses, it is not clear whether

DPI administered at ultra-low dose is still capable

of inhibiting NADPH oxidase activation in vivo. To

address this question, we determined the effects of

Figure 2 Post-treatment with an ultra-low dose of DPI protects dopaminergic neurons against LPS-induced damage in

transgenic mice over-expressing human A53T mutant a-synuclein. (A) Experimental designs. Seven-month-old transgenic mice over-expressing human A53T mutant -synuclein received a single injection of LPS [6 106 EU/kg, intraperitoneally (i.p.)]. One month after LPSinjection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. (B) Three months

after DPI post-treatment, nigral dopaminergic neurons and striatal axon fibres were immunostained with anti-tyrosine hydroxylase antibody, and

representative images are shown. (C) The number of tyrosine hydroxylase-immunoreactive cells was counted stereologically, and the results are

expressed as the mean SEM. (D) The density of tyrosine hydroxylase immunostaining in the striatum was quantified using densitometricanalysis. Data were analysed by one-way ANOVA followed by Tukeys post hoc testing. **P5 0.01; Scale bar = 200 mm; n = 56. TH = tyrosinehydroxylase.

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ultra-low dose DPI on reactive oxygen species production

in the brain after 10 months of LPS injection (Fig. 5A). Our

previous report showed that a single systemic LPS injection

causes persistent NADPH oxidase activation and related

reactive oxygen species production in the mouse brain

(Qin et al., 2013). Electron spin resonance analysis further

conrmed that NADPH oxidase, but not other oxidases,

such as xanthine oxidase, accounts for the major

(4 90%) source of LPS-induced oxidative stress in vivo(Sato et al., 2002). In situ visualization of reactive

oxygen species production was performed using dihy-

droethidium, a reactive oxygen species-sensitive dye.

Dihydroethidium can readily cross the bloodbrain barrier

and exhibits red uorescence through interactions with

superoxide and other free radicals in the brain (Wu et al.,

2003). As shown in Fig. 5B, reactive oxygen species pro-

duction in the substantia nigra was minimal in vehicle

controls, as evidenced by the low levels of red uorescence.

In contrast, exposure to LPS resulted in increased levels of

red uorescence in the substantia nigra, indicating elevated

reactive oxygen species production. Co-staining with tyro-

sine hydroxylase antibody revealed a high degree of oxida-

tive stress in the dopaminergic neurons of LPS-injected mice

(Fig. 5C). Post-treatment with ultra-low dose DPI markedly

reduced LPS-induced oxidative stress in the nigra

and nigral dopaminergic neurons (Fig. 5B and C).

Quantitative analysis revealed 2.3-fold increase in uores-

cence density in LPS-injected mice compared with vehicle

controls, which was reduced to 1.1-fold in DPI/LPS-treated

mice (P5 0.0001; Fig. 5D). The inhibitory effects of DPIon LPS-induced reactive oxygen species production suggest

that ultra-low dose DPI can inhibit NADPH oxidase

activation.

The source of reactive oxygen species production has

recently become a subject of debate. Mitochondria have

been traditionally considered a major source of intracellular

reactive oxygen species production; however, our previous

nding indicates that NADPH oxidase is a key reactive

oxygen species-generating enzyme in microglia in response

to LPS stimulation (Qin et al., 2013). In addition to

NADPH oxidase, DPI at regularly used doses (between 1

to 5mg/kg) inhibits a variety of electron-transferring avo-

protein enzymes, including mitochondrial complex

I (Aldieri et al., 2008). To investigate whether ultra-low

dose DPI has specicity toward NADPH oxidase in vivo,

we evaluated the effects of DPI on complex I activity.

Interestingly, DPI at 10 ng/kg failed to suppress the activ-

ities of mitochondrial complex I in the brain (Fig. 5E). This

result indicated a high specicity of ultra-low dose DPI in

inhibiting NADPH oxidase and revealed a critical role

of this superoxide-producing enzyme in the generation of

reactive oxygen species in the brain.

Figure 3 Post-treatment with an ultra-low dose of DPI attenuates nigral a-synuclein aggregation in LPS-treated transgenicmice over-expressing human A53T mutant a-synuclein. (A) One month after LPS injection, transgenic mice over-expressing human A53Tmutant -synuclein were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for 2 weeks. Three months

after DPI post-treatment, human -synuclein was immunostained in the substantia nigra with SYN211 (specific for human -synuclein) antibody,

and representative images are shown. (B) Magnifications of dopaminergic neuron (tyrosine hydroxylase-immunoreactive) and -synuclein double

staining are indicated in the different groups. (C) The SYN211 density in the substantia nigra was quantified. The results are expressed as a

percentage of the vehicle controls (mean SEM) and were analysed by one-way ANOVA followed by Tukeys post hoc testing. **P5 0.01; Scalebar = 200 mm in (A) and 50mm in (B); n = 56. -Syn = -synuclein.

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Ultra-low dose DPI affords long-termdopaminergic neuroprotectionagainst MPTP lesions

In addition to using LPS models, we tested the protective

effects of ultra-low dose DPI in a MPTP Parkinsons disease

mouse model, which has been widely used to screen for

therapeutic agents. Unlike LPS, MPTP directly damages

dopaminergic neurons in an acute fashion. Therefore, we

initially evaluated the neuroprotective potential of DPI

using a pre-treatment regimen. DPI infusion (10 ng/kg/

day, subcutaneously) started 2 days before MPTP adminis-

tration and lasted for 2 weeks (Supplementary Fig. 2A).

The neuroprotective effects of DPI were evaluated at differ-

ent time points (27, 60 and 120 days after the initial

injection of MPTP). The chosen 27-day point was based

on our previous report (Hu et al., 2008) showing nearly

50% loss of dopaminergic neurons in the substantia nigra

pars compacta. Consistently, MPTP elicited a 45% loss of

tyrosine hydroxylase-immunoreactive neurons in the sub-

stantia nigra pars compacta compared with vehicle controls

at the 27-day point. The loss of nigral tyrosine hydroxy-

lase-immunoreactive neurons remained close to the same

degree at the 60- and 120-day time points. MPTP-elicited

tyrosine hydroxylase-immunoreactive neuronal loss was

signicantly attenuated by DPI (P = 0.0032, compared

with the MPTP alone group) at the 27-day time point.

The long-lasting protective effect of DPI was evident

based on the results obtained at the 60- and 120-day

time points (Supplementary Fig. 2B). To exclude the

Figure 4 Post-treatment with an ultra-low dose of DPI attenuates chronic microglial activation. (A) Three (pre-motor) or 10

(motor) months after LPS injection, mice (C57BL/6J) were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-

pump for 2 weeks. Two microglial markers, ITGAM (CD11b) or AIF1 (Iba-1), were immunostained in the substantia nigra region 7 months after

DPI treatment. Representative pictures of staining are shown. Activated microglia characterized by an enlarged cell body size and high staining

density. (BD) The activation of microglia was quantified by measuring the density of ITGAM (CD11b; B) and AIF1 (Iba-1) (C) and the cell body

size (D). The gene expressions of tumor necrosis factor alpha (TNF; E), interleukin-1 beta (Il-1b; F) and major histocompatibility complex II (G)in brain were determined in the rostral half of the brains using RT-PCR. Data are expressed as a percentage of time-matched vehicle controls

(mean SEM) and were analysed by two-way ANOVA followed by Tukeys post hoc testing. *P5 0.05, **P5 0.01; n = 56; Scale bar = 50 mm.MHCII = major histocompatibility complex II.

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possibility that DPI-afforded protection was due to either

alterations of MPTP metabolism or entry into the brain, we

measured MPP+ levels in the brain after MPTP injection

and found no signicant difference between MPTP and

DPI/MPTP (data not shown).

In addition to evaluating the DPI-afforded protection of

nigral tyrosine hydroxylase-immunoreactive neurons, stri-

atal levels of dopamine and its metabolite, DOPAC as

well as their turnover rate (DOPAC/dopamine) were

measured as markers for the functional recovery of dopa-

minergic neurons (Zigmond et al., 1990; Zigmond, 1997).

Twenty-seven days after the MPTP injections, marked de-

creases in striatal levels of dopamine (68%) compared with

the vehicle controls were observed (Supplementary Fig.

2C); in contrast, the decrease in DOPAC level was attenu-

ated (Supplementary Fig. 2D). The higher ratio of DOPAC/

dopamine in the MPTP-treated group further indicated

a higher turnover of dopamine, likely due to a compensa-

tory effect of dopaminergic neuron loss (Supplementary

Fig. 2E). Interestingly, whereas DPI did not prevent the

MPTP-induced loss of striatal dopamine levels, signicantly

high levels of DOPAC (P = 0.0207, compared with the

MPTP alone group) and DOPAC/dopamine ratios

(P = 0.0284 compared with the MPTP alone group) were

observed in the DPI-treated group (Supplementary Fig. 2D

and E), suggesting enhanced functional activities of the

surviving dopaminergic neurons (Zigmond et al., 1990;

Zigmond, 1997). A time-dependent recovery of dopamine

levels was found at the 60-day time point for both the

MPTP/vehicle and the MPTP/DPI groups compared with

the levels at the 27-day time point. Although the striatal

levels of dopamine were the same between these two

groups, the DOPAC level and DOPAC/dopamine ratio re-

mained high in the DPI-treated group, suggesting that DPI

preserved some degree of functional activity in remaining

dopaminergic neurons. Continuing recovery of striatal

dopamine levels was shown at the 120-day time point in

the MPTP-treated group. It is interesting to note that the

levels of dopamine in DPI/MPTP-treated mice returned to

the same level as non-MPTP-injected mice (P = 0.9960;

Figure 5 Post-treatment with an ultra-low dose of DPI inhibits the LPS-induced increase in dihydroxyethidium oxidation but

not the activity of mitochondrial complex I. (A) Experimental designs. Ten months after LPS injection, mice (C57BL/6J) were infused with

DPI (10 ng/kg/day). After 2 weeks of DPI infusion, the brains were perfused, and the production of superoxide was determined by measuring the

oxidation products of dihydroethidium by fluorescence microscopy. (B) Representative images of dihydroethidium staining (red) are shown. The

position of the substantia nigra was identified by tyrosine hydroxylase staining (green). (C) Magnification of dopaminergic neurons (tyrosine

hydroxylase-immunoreactive) and dihydroethidium double staining are indicated in the different groups. (D) Quantified data showing the fluor-

escence density of the dihydroethidium oxidation in the substantia nigra of different treatment groups. Fluorescence density of NADPH oxidase-

deficient (gp91phox /) mice served as a control background. (E) The effects of ultra-low-dose DPI on the activities of complex I in the brain weredetermined using commercial assay kits. Data are expressed as a percentage of time-matched vehicle controls (mean SEM) and were analysedby Students t-test (E) or one-way ANOVA followed by Tukeys post hoc testing (D). **P5 0.01; n = 5; Scale bar = 100 mm.DHE = dihydroethidium; TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.

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Supplementary Fig. 2C). The rapid recovery of striatal

dopamine levels supported the promising neuroprotective

effects of DPI.

Ultra-low dose DPI attenuatesMPTP-induced oxidative stress andmicroglial activation

Additional studies were performed to elucidate the mech-

anism by which DPI protected against MPTP-elicited

neurotoxicity. Although MPTP is directly toxic to dopa-

minergic neurons, microglia-mediated neuroinammation

contributes to its overall neurotoxicity (Wu et al., 2003;

Hu et al., 2008; Levesque et al., 2010). Microglia are

indirectly activated to produce cytotoxic factors (reactive

microgliosis) in response to MPTP-induced neuronal

damage, causing additional dopaminergic degeneration

(Levesque et al., 2010). To provide evidence that microglia

play a role in DPI-elicited neuroprotection, immunostaining

of microglial markers was performed. DPI treatment signi-

cantly attenuated MPTP-induced microglial activation,

as shown by the reduced cell body size and densities of

ITGAM and AIF1 staining in the substantia nigra

(Supplementary Fig. 3A). Quantitative analysis revealed

decreased immunoreactivities of ITGAM (P = 0.0466) and

AIF1 (P = 0.0385) in the substantia nigra compared with

the MPTP alone group (Supplementary Fig. 3B). Moreover,

DPI signicantly reduced MPTP-elicited oxidative stress

in the substantia nigra based on the low nigral density

of dihydroethidium oxidation (red uorescence) in the

DPI/MPTP group compared with the MPTP alone group

(P = 0.0039; Supplementary Fig. 4A and C). Consistent

with the LPS model, co-staining with tyrosine hydroxylase

antibody revealed that DPI treatment reduced oxidative

stress in dopaminergic neurons (Supplementary Fig. 4B).

Overall, our results show that ultra-low dose DPI

inhibits MPTP-induced reactive microgliosis and oxidative

stress.

NADPH oxidase deficiency abolishesultra-low dose DPI-affordedneuroprotection

To investigate whether NADPH oxidase mediates ultra-

low dose DPI-afforded neuroprotection, NADPH oxi-

dase-decient (gp91phox /) and wild-type control(gp91phox+ / + ) mice were treated with DPI followed by le-

sioning with MPTP. Consistent with the data in

Supplementary Fig. 2B, in wild-type mice, MPTP injection

caused a 46% loss of nigral tyrosine hydroxylase-

immunoreactive neurons and had only 23% loss in DPI/

MPTP-treated group (P = 0.0001; Supplementary Fig. 5A

and C), compared to the vehicle control. In contrast, mice

lacking gp91phox were more resistant to MPTP-induced

lesions than wild-type controls (27 3% versus 46 3%loss of nigral tyrosine hydroxylase-immunoreactive

neurons, P = 0.0027), supporting the potential involvement

of NADPH oxidase in MPTP-induced dopaminergic neuron

damage. Under this condition, ultra-low dose DPI failed to

protect nigral dopaminergic neurons in gp91phox / micewith MPTP lesions (P = 0.9997, compared with MPTP

alone group). Consistently, reduced degeneration of ter-

minals of dopaminergic neurons in the striatum were

observed in DPI/MPTP-treated wild type (gp91phox\ + )

mice, but no difference was detected in NADPH oxidase-

decient (gp91phox /) mice (Supplementary Fig. 5Band D).

Post-treatment with DPI attenuatesMPTP-induced dopaminergicdegeneration and motor deficits

The positive results from the pretreatment studies

(Supplementary Fig. 2 to 5) led us to explore the possibility

of a post-treatment regimen to further assess the thera-

peutic efcacy of ultra-low dose DPI in the MPTP model.

DPI (10 ng/kg/day) was post-administered at Day 3 after

the rst injection of MPTP for 2 weeks (Fig. 6A). Pilot

studies showed a 2030% loss of nigral tyrosine hydro-xylase-immunoreactive neurons when DPI infusion

was initiated. Twenty-seven days after MPTP lesion, a

signicantly greater number of nigral tyrosine

hydroxylase-immunoreactive neurons was found in DPI/

MPTP-treated mice (P = 0.0239, compared with the

MPTP alone group; Fig. 6B). Consistent with the pretreat-

ment regimens, DPI post-treatment enhanced the activity of

nigral dopaminergic neurons, as shown by an increased

dopamine turnover rate compared to the MPTP alone

group (P = 0.0067; Fig. 6C). The neuroprotective effect of

DPI post-treatment was correlated with the attenuation of

MPTP-elicited motor decits (P = 0.0470), as measured by

the rotarod test (Fig. 6D). Longer-term studies similar to

Supplementary Fig. 2 were not conducted in this post-treat-

ment regimen. However, based on the high turnover rate of

dopamine and improved motor behaviour in DPI-treated

mice, it is likely that mice post-treated with DPI would

display an accelerated recovery from MPTP treatment at

later time points.

Ultra-low dose DPI displays no overttoxicity in mice

The severe toxicity of DPI at the recommended dose ham-

pers its potential for clinical usage in patients. Thus, we

conducted a toxicological screen of DPI when administered

at ultra-low dose. All parameters studied in this project,

including the quantitative and morphological analysis

(neurons and microglia), neurochemical measurements

(proinammatory factors, complex I activity and dihy-

droethidium oxidation) and behavioural observations

(rotarod activities, Fig. 7A), showed no difference in DPI

alone group compared to vehicle controls. General

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evaluation of major organ systems and organ weights

concluded that ultra-low dose DPI treatment had no signi-

cant effects on body and organ weights (including liver,

kidney and spleen; Fig. 7BE) nor any observable histo-

pathological changes in haematoxylin and eosin-staining

of sections from liver, spleen, kidney, testes, heart and

lung (Fig. 7F) when compared to saline treated controls.

Additionally, haematoxylin and eosin staining of brain sec-

tions based on the modied National Toxicology Program

protocol (Rao et al., 2013) also revealed no signicant

changes in DPI-treated mice compared to vehicle controls

(Fig. 7F). We further determined whether low doses of DPI

affect the peripheral immune function by examining both

high and low doses of DPI on superoxide production in

mice neutrophils. Results indicated that high dose DPI

(105M) completely inhibited superoxide production, butDPI in low concentrations (1013M and 1014M) failed toaffect the oxidative burst of neutrophils (Supplementary

Fig. 6). Our preliminary data also showed no effect of

low-dose DPI on the superoxide production of human

neutrophils (data not shown). Overall, these ndings sug-

gest that ultra-low dose DPI displayed no overt organ tox-

icity and does not inuence peripheral immune cell

functions that are critical for hosting normal immune

responses.

DiscussionThe present study demonstrated that DPI at an ultra-low

dose provides potent benecial effects in three models of

dopaminergic degeneration. One salient feature was the

high efcacy of DPI in neuroprotection, even when admin-

istered in a post-treatment regimen after the onset of dopa-

minergic neuron damage (see summary in Supplementary

Table 2). Our results strongly support that the neuropro-

tective effects of ultra-low dose DPI occur by specically

inhibiting NADPH oxidase and subsequently reducing

microglia-mediated chronic neuroinammation (Fig. 8).

Additionally, mice treated with ultra-low dose DPI

showed no overt signs of toxicity. The efcacy of a post-

treatment regimen in chronic progressive dopaminergic

neuron degenerative models together with its low toxicity

suggest that ultra-low dose DPI may be a promising drug

candidate for future human studies.

Over the past decade, the development of drugs capable

of modifying disease progression in Parkinsons disease has

been unsuccessful. Despite the encouraging results reported

in numerous animal studies, a small percentage of these

compounds have been tested in clinical trials with even

fewer reaching the clinic (Hart et al., 2009; Brichta et al.,

2013). One particular failure in translating these drugs has

Figure 6 Post-treatment with an ultra-low dose of DPI attenuates MPTP-induced dopaminergic neuron damage and motor

deficits. (A) Experimental designs. Repeated MPTP regimens (15 mg/kg, subcutaneously for 6 consecutive days) were administered to C57BL/6J

mice. After 3 days of MPTP injection, the mice were infused with either vehicle or DPI (10 ng/kg/day; subcutaneously) via osmotic mini-pump for

2 weeks. (B) Twenty-seven days after the first injection of MPTP, dopaminergic neurons in the substantia nigra pars compacta were immunos-

tained with anti-tyrosine hydroxylase antibody, and the numbers of tyrosine hydroxylase-immunoreactive cells were counted. (C) The turnover

rate of dopamine in the striatum was calculated by the ratio of dopamine metabolite (DOPAC) and dopamine. (D) The protective effects of DPI

against MPTP-induced motor deficits were measured by the rotarod test. Data are expressed as the mean SEM and were analyzed by one-wayANOVA followed by Tukeys post hoc testing. *P5 0.05, **P5 0.01; n = 810. TH = tyrosine hydroxylase; THir = tyrosine hydroxylase-immunoreactive.

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been the lack of suitable Parkinsons disease models for

drug development, which prompted us to create two

inammation-based progressive neurodegenerative models

by using a systemic injection of LPS to either C57BL/6J

or transgenic mice over-expressing human A53T mutant

-synuclein (Qin et al., 2007; Gao et al., 2011). These

new models improve upon existing models by recapitulat-

ing the delayed and progressive degeneration of nigrostria-

tal dopaminergic neurons (Qin et al., 2007; Gao et al.,

2011), showing temporary recovery of motor decits by

L-DOPA (Qin et al., 2007; Liu et al., 2008) and generating

-synuclein-positive inclusion bodies in the substantia nigra

(Liu et al., 2008; Gao et al., 2011). Administering an ultra-

low dose of DPI to these mouse models not only halted the

progression of neurodegeneration in mice that already

exhibited more than a 30% loss of nigral dopaminergic

neurons but also attenuated -synuclein accumulation in

dopaminergic neurons in the transgenic mice over-express-

ing human A53T mutant -synuclein (Fig. 3). To our

knowledge, this is the rst report demonstrating a therapy

capable of halting progressive dopaminergic neurodegen-

eration, reducing -synuclein accumulation, attenuating

the depletion of striatal dopamine, and improving motor

behaviours even when administered as a post-degenerative

intervention.

Mechanistically, the most critical question to address

is why such a low dose of DPI post-administered at

the motor stage of disease progression still displays neuro-

protection and reverses motor decits. The results of

the present study, together with our previous reports

(Block et al., 2007; Gao and Hong, 2008; Qin et al.,

2013), suggest that DPI achieves these extraordinary pro-

tective effects through the inhibition of NADPH oxidase

and subsequent interruption of microglia-mediated chronic

neuroinammation. Although microglial activation is essen-

tial to restore brain homeostasis after an injury or infection

(Streit, 2000), it may become pathological if the initial in-

ammation is not properly resolved. Release of noxious

endogenous ligands generated by injured neurons, such as

m-calpain, -synuclein and high mobility group box 1, arethought to continually reactivate microglia (reactive micro-

gliosis) resulting in additional neurodegeneration (Block

et al., 2007; Levesque et al., 2010; Gao et al., 2011).

Consequently, a self-propelling vicious cycle is created

through interactions between injured neurons and dysregu-

lated microglia, inevitably resulting in the delayed and pro-

gressive collateral neurodegeneration of dopaminergic

neurons in Parkinsons disease (Gao and Hong, 2008).

We previously recognized NADPH oxidase as a key medi-

ator in bridging chronic neuroinammation and progressive

dopaminergic neurodegeneration (Qin et al., 2004; Zhang

et al., 2004; Block et al., 2007; Gao and Hong, 2008). This

nding led us to theorize that inhibiting NADPH oxidase

could effectively disrupt this self-propelling vicious cycle,

potentially resulting in a new disease-modifying strategy

for Parkinsons disease. As predicted, ultra-low dose DPI

effectively reduced LPS-induced oxidative stress, which was

mainly derived from NADPH oxidase activation and sub-

sequent superoxide production (Fig. 5 and Supplementary

Fig. 4). The inhibition of NADPH oxidase not only reduced

Figure 7 An ultra-low dose of DPI displays no toxicity. Mice were treated with either vehicle or DPI (10 ng/kg/day; subcutaneously) using

an Alzet mini-pump for 2 weeks. The effects of ultra-low-dose DPI on rotarod (A), and body (B), liver (C), spleen (D) and kidney (E) weights

were assessed. (F) Organs including liver, spleen, kidney, testis, heart, lung and brain were dissected and stained with haematoxylin and eosin.

Representative images are shown. Data were analysed using a Students t-test. n = 3; Scale bar = 100 mm.

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extracellular superoxide but also decreased intracellular

reactive oxygen species levels thought to be important sec-

ondary messengers that regulate the expression of many

proinammatory factors by activating several downstream

signalling pathways including protein kinase C, mitogen-

activated protein kinase and NF-B (Block and Hong,

2005). Consistent with this mode-of-action, post-treatment

with DPI produced long-term inhibition of LPS-elicited

microglial activation and suppressed gene expression of

proinammatory factors tumor necrosis factor alpha and

interleukin-1 beta (Fig. 4). Unlike conventional anti-inam-

matory therapies that directly suppress certain pro-inam-

matory factors, selective inhibition of NADPH oxidase by

DPI can inactivate microglia and prevent the generation of

a spectrum of pro-inammatory factors. Although this

study focused primarily on microglial NADPH oxidase,

neurons are also express this superoxide-producing

enzyme at much lower quantities and thus, we cannot ex-

clude the possibility that the inhibition of neuronal

NADPH oxidase might also contribute to the DPI-elicited

neuroprotection.

In addition to using LPS models, the efcacy of

DPI was demonstrated in a more conventional MPTP

model. Although previous studies have demonstrated pro-

tection by post-treatment with rottlerin (protein kinase C

inhibitor) (Zhang et al., 2007) or caffeine (Xu et al., 2010)

in a MPTP mouse model, their protective effects were only

observed when the drugs were administered within a couple

of hours after MPTP lesion. By using a subchronic MPTP

model, we showed that DPI (10 ng/kg/day) post-treatment

after 3 days of initial MPTP injection attenuated dopamin-

ergic neurodegeneration and improved rotarod activity.

It is interesting to compare the LPS and MPTP models in

this study. LPS is known to directly activate microglia and

trigger neuroinammation to produce delayed and progres-

sive nigral neurodegeneration (Qin et al., 2007). In con-

trast, MPTP causes acute dopaminergic neurotoxicity by

inhibiting mitochondrial complex I. The different modes-

of-action of these models begs the question how DPI can

partially prevent dopaminergic neuronal loss, improve

dopamine turnover and restore motor function in a

model of direct neuronal lesion (Fig. 6). Although MPTP

cannot directly activate microglia, stress signals released by

dying neurons derive reactive microgliosis to generate

superoxide via NADPH oxidase (Block et al., 2007;

Levesque et al., 2010). We and others have previously

reported that reactive microgliosis generated in response

to the acute lesioning of dopaminergic neurons by MPTP

trigger a delayed collateral neurotoxicity from neuroinam-

mation after the acute phase of MPTP (Gao et al., 2003;

Wu et al., 2003; Hu et al., 2008; Levesque et al., 2010).

Thus, we believe that DPI protected against this delayed

toxicity by attenuating reactive microgliosis through the

inhibition of NADPH oxidase activity.

It is important to note that although DPI potently inhibits

microglial activation in both LPS and MPTP models, DPI

alone did not produce microglial cytotoxicity nor did it sup-

press the basal microglial activity as illustrated by the

similar densities of AIF1 and ITGAM-positive microglia

and the gene expressions of proinammatory cytokines

Figure 8 Proposed model showing how ultra-low-dose DPI attenuates progressive dopaminergic neurodegeneration. NADPH

oxidase is a key mediator for initiating and maintaining the self-propagating vicious cycle formed between damaged neurons and dysregulated

microglia. The self-propelling vicious cycle is critical in driving the progressive dopaminergic degeneration in Parkinsons disease. Ultra-low dose

DPI is capable of inhibiting the activation of NADPH oxidase and subsequent production of superoxide and other neurotoxic factors to mitigate

chronic neuroinflammation. Once the self-propelling vicious cycle is interrupted by inhibiting NADPH oxidase on microglia, neurons or both, the

progression of dopaminergic neuron degeneration can be halted. These results provide a novel and promising avenue for developing drug therapy

for Parkinsons disease and other neurodegenerative diseases.

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in comparison with the vehicle controls (Fig. 4). This

observation is clinically relevant because DPI attenuates

only the induced microglial activation without interfering

with normal microglial immune surveillance function.

Taken together, our ndings suggest that the reduction

of chronic microglia-mediated neuroinammation through

the inhibition of NADPH oxidase is an effective strategy

to halt disease progression in both inammation (LPS)- and

neurotoxin (MPTP)-based rodent Parkinsons disease

models.

This study also addressed some critical issues relevant to

the potential clinical use of ultra-low dose DPI in future

human clinical trials. The rst issue was resolving the dur-

ation of treatment necessary to achieve an observable

effect. To our surprise, we found that the subcutaneous

infusion of DPI for two weeks was sufcient to provide

potent neuroprotection. Although it is not clear why

long-term administration was not necessary, one possible

explanation for this effect is that once the self-propelling

vicious cycle is interrupted by DPI, chronic neuroinamma-

tion ceases unless additional inammation is induced in the

brain. The second issue was whether DPI could specically

inhibit NADPH oxidase in vivo. In micromolar concentra-

tions, DPI inhibits several essential cytochrome-containing

enzymes beyond just NADPH oxidase, attributing to its

high cytotoxicity at these concentrations (Gatley and

Martin, 1979; Aldieri et al., 2008). We recently reportedthat subpicomolar concentrations of DPI have great

specicity to potently inhibit NADPH oxidase-generated

superoxide without affecting the activities of other cyto-

chrome-containing enzymes, such as inducible nitric oxide

synthase, xanthine oxidase, cytochrome P450 reductase,

thioredoxin reductase, and complex I, in cultured microglia

(Wang et al., 2014a). Although the exact brain concentra-

tion of DPI in our in vivo studies was too low to beaccurately measured, data extrapolation from a previous

pharmacokinetic study (Gatley and Martin, 1979) estimate

that the brain DPI levels in treated mice were similar to the

range (1014 to 1013M) used in the aforementionedin vitro studies (Wang et al., 2014a). The specicity of

this ultra-low dose DPI in vivo was further supported by

showing no changes of brain mitochondrial complex I ac-

tivity in the DPI-treated mice (Fig. 5).

Another critical issue was whether ultra-low dose DPI

could generate toxicity in vivo. To examine this, we con-

ducted a standard pathological evaluation based on the

modied National Toxicology Program/National Institutes

of Health protocol. This method measures the weight of the

body and organs, behavioural activity, and performs histo-

logical assessment of organs tissues to verify the safety

prole of putative toxicants. Although these methods

cannot rule out subtle toxicities, no gross toxicological ef-

fects were noted. Furthermore, as we know that patients

with chronic granulomatous disease (CGD), a rare muta-

tion on NADPH oxidase that render the subunit functional

inactive, and NADPH oxidase decient mice display im-

munodeciency, we conrmed that ultra-low dose DPI

did not affect the peripheral immune cell functions in

both mice (Supplementary Fig. 6) and human (data not

shown) that are critical for hosting normal immune

responses. Finally, it is important to point out that al-

though we provided strong evidence for the potential clin-

ical usage of ultra-low dose DPI in Parkinsons disease,

there still have several issues to consider before translating

our ndings. First, in our study, the DPI infusions are given

when about 3035% of dopaminergic neurons are lost. In

humans, the degree of nigral dopaminergic neuron loss is

much greater (about 5060% loss) (Hirsch, 2007) than this

when the patients rst show motor symptoms. It remains to

be determined whether DPI treatment is still effective in

Parkinsons disease patients, who already display motor

symptoms. Second, our study showed no overt toxicity in

low-dose DPI-treated mice; however, a more detailed evalu-

ation for the toxicity of low-dose DPI in monkeys or even

human is needed. Third, although neuroinammation has

been recognized as one of the critical factors that contribute

to the progression of Parkinsons disease, the complexity of

disease aetiology makes it difcult to predict whether DPI

could work as effectively in human Parkinsons disease

patients.

In summary, this study provides convincing evidence that

subchronic infusion of an ultra-low dose of DPI potently

reduced microglia-mediated chronic neuroinammation by

selectively inhibiting NADPH oxidase and halted progres-

sive neurodegeneration in both LPS and MPTP models.

Our ndings may provide a novel and efcient therapeutic

strategy for future Parkinsons disease therapy. The ability

to halt progressive neurodegeneration, selective specicity

in inhibiting NADPH oxidase and initial safety proles sug-

gest that ultra-low dose DPI could be a promising candi-

date for future clinical trials in patients with Parkinsons

disease.

FundingThis research was supported by the Intramural Research

Program of the NIH, National Institute of Environmental

Health Sciences

Supplementary materialSupplementary material is available at Brain online.

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