Neuroinflammation and oxidative stress are now well recognized as key pathophysiological events contributing to the progressive loss of nigral dopaminergic neurons in PD [2–4]. However, an effective neuroprotective therapy to halt the progression of the disease is not available. Here, we report the anti-inflammatory and antioxidative properties of a synthetic analog of apocynin in the MPTP mouse model of PD. Recent studies have shown conversion of apocynin to diapocynin (apocynin dimer) in vivo, which prevents the assembly and activation of the NADPH oxidase complex . Additionally, diapocynin is 13-fold more lipophilic than apocynin . Here, we show that diapocynin inhibits MPTP-induced activation and expression of both iNOS and gp91phox in activated glial cells, suggesting that diapocynin has anti-inflammatory properties against neurotoxic stress. Diapocynin also attenuates the formation of ONOO- and 4-HNE in dopaminergic neurons in response to various stimuli, further confirming the antioxidant properties of this compound.
Importantly, diapocynin also protects against MPTP-induced motor deficits, striatal neurotransmitter depletion and nigrostriatal degeneration. Furthermore, diapocynin is effective in post-treatment paradigms as well as in chronic neurodegenerative models of PD. Derivatives of natural compounds, such as diapocynin, are a key translational approach for the development of therapies for PD. To our knowledge, this is the first report showing anti-inflammatory, antioxidative and neuroprotective properties of a novel apocynin derivative in animal models of PD.
NADPH oxidase has emerged as a major source of oxidative stress in the brain, particularly in neurodegenerative disorders, such as PD, Alzheimer's disease, ALS and multiple sclerosis . Apocynin has been shown to inhibit NADPH oxidase, which generates ROS during inflammatory processes . Although the mechanism of inhibition of apocynin is not clear, it is thought to prevent the recruitment of cytosolic NADPH oxidase subunit p47phox to the membrane, thereby inhibiting NADPH oxidase activity. Apocynin has been shown to attenuate superoxide formation and oxidative stress in vivo as well as reduce acute inflammation in lung and spinal cord [24–26]. Furthermore, apocynin administered at a dose of 300 mg/kg/day protects against oxidative damage induced by cerebral ischemia  and ALS .
In contrast, recent studies have shown that apocynin failed to show any improvement in transgenic animal models of Alzheimer's disease  or ALS . In vitro studies in dopaminergic neuronal cell lines and primary cultures also demonstrated a protective role of apocynin in 1-methyl-4-phenyl-pyridinium ion (MPP+) or MPTP-induced NADPH oxidase mediated apoptotic cell death [10, 30]. Also, a pro-oxidative nature of apocynin has been shown in non-phagocytic cells, where it increases ROS production significantly . Thus, these studies suggest that the development of an improved apocynin related compound may yield a better neuroprotective agent for treatment of PD.
In the central nervous system, glial activation involving astrocytes, microglial cells, lymphocyte infiltration, and production of proinflammatory mediators including cytokines, chemokines, prostaglandins, and reactive mediators, such as reactive nitrogen species (RNS) and ROS, are all hallmarks of inflammatory reactions. MPP+, the active metabolite of MPTP, is believed to be responsible for glial activation mediated inflammation and neurodegeneration . In our study, we also observed marked activation of microglia and astrocytes, measured by Western blotting and immunohistochemistry after MPTP treatment in SN, and diapocynin significantly attenuated MPTP-induced microgliosis and astrogliosis (Figure 2).
Nuclear factor kappa B (NF-κB), a transcription factor, has been shown to be an important regulator of the microglial and astroglial proinflammatory reactions in the SN. The promoter regions of proinflammatory molecules, including iNOS, contain the binding sites for NF-ĸB . Astroglia and microglia in the healthy brain do not express iNOS, but following toxic or inflammatory damage, reactive astroglia and microglia express iNOS in the brain . Studies have shown that MPTP treatment produces significantly reduced neuronal loss in mice deficient in iNOS compared to their wild-type counterparts .
In this study, we demonstrate that diapocynin, a pharmacological inhibitor of microglial NADPH oxidase, effectively attenuates MPTP-induced increases in iNOS expression (Figure 3), suggesting the potential use of diapocynin as an anti-inflammatory agent. RNS as well as ROS play a pivotal role in oxidative stress and inflammation in PD. NADPH oxidase, which is a major ROS-producing enzyme of microglial cells, mediates superoxide production and controls the levels of pro-inflammatory neurotoxic factors, such as TNFα and IL-1β . In our study, we demonstrate that diapocynin attenuates MPTP-induced expression of microglial gp91phox in SN and thereby reduces the production of ROS (Figure 4).
Besides having direct toxic effects on nigral dopaminergic neurons, nitric oxide (·NO) and superoxide O2− derived from astrocytes and microglia can react to form the highly reactive nitrogen species peroxynitrite (ONOO-). Peroxynitrite causes nitration of tyrosine residues in various proteins including TH and α-synuclein [21, 35]. Peroxinitrite mediated nitration of TH is associated with reduced enzymatic activity.
3-NT is widely used as a marker of nitrative damage. Here, we found increased expression of 3-NT in dopaminergic neurons in SN of MPTP-treated mice, predominantly co-localized with TH-positive dopaminergic neurons (Figure 5A,B,C). However, diapocynin significantly decreased the MPTP-induced increase in 3-NT in dopaminergic neurons in the SN.
Along with peroxynitrite, levels of 4-HNE, an unsaturated aldehyde generated during lipid peroxidation, were also significantly increased in the SN of PD brains compared to controls . 4-HNE has been demonstrated to block mitochondrial respiration and induce caspase-dependent apoptosis [37, 38]. In our study, we showed increased expression of 4-HNE, a marker of oxidative damage in the SN of MPTP-treated mice, which was colocalized in the cytosol of TH-positive dopaminergic neurons (Figure 5D,E,F). However, diapocynin significantly decreased the amount of 4-HNE in the MPTP-treated SN, indicating that diapocynin acted by attenuating ROS generation.
The lack of an effective therapy to halt the progression of PD has been a longstanding challenge in the field. Administration of a dopamine agonist or levodopa has been the leading treatment for PD symptoms, but these treatments do not affect disease progression. Various putative neuroprotective agents, including glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), TGF-β and other small molecule compounds, have been tested in animal models of PD [39, 40]. However, most of these compounds failed in either pre-clinical trials or human trials due to their inability to cross the blood-brain barrier or due to limited bioavailability. Moreover, they also caused adverse side effects. Hence, understanding the mechanism of the disease process and development of a successful neuroprotective therapeutic approach to halt the disease progression are of principal importance in PD research.
Diapocynin has several advantages compared to other experimental drugs, including its parent compound apocynin. These include: (1) co-treatment of diapocynin and MPTP profoundly attenuated MPTP-induced glial activation and proinflammatory events, (2) diapocynin suppressed oxidative stress in vivo in the SN of MPTP-treated mice, (3) diapocynin treatment improved MPTP-induced behavioral deficits, (4) diapocynin protected TH-positive dopaminergic neurons from MPTP toxicity and restored the level of dopamine and its metabolites, and (5) oral administration of diapocynin on day 4, after the disease has been initiated by MPTP, also restored the levels of striatal dopamine neurotransmitters in MPTP-treated mice, suggesting that diapocynin could attenuate disease progression.
It is noteworthy that diapocynin does not interfere with MPTP metabolism, demonstrating the true neuroprotective effect in the MPTP model. Also, diapocynin is fairly nontoxic, as the mice treated with diapocynin alone (300 mg/kg/day) for 12 days did not show any sign of behavioral imparities and their neurotransmitter levels were not different from the saline-treated control mice (Figures 1E,F,G,H and 8D,E,F). Another advantage is that diapocynin can be administered orally by gavage. Being a lipophilic molecule, diapocynin easily crosses the blood-brain barrier and enters the SN (>1.5 μg/mg tissue) and striatum (>0.9 μg/mg tissue) regions of brain, as detected by LC/MS/MS (Figure 1C and D). We had to use 300 mg/kg oral dose in order to achieve a desired neuroprotective effect. Although the exact reason for the requirement of a high dose of diapocynin is not clear, it is possible that diapocynin rapidly undergoes metabolic degradation similar to apocynin . Nevertheless, future studies are needed to clarify the metabolic fate of diapocynin in vivo. Taken together, our results demonstrate that diapocynin is a promising neuroprotective agent that deserves further exploration for its use in clinical settings.