Sex-based disparity in paraoxonase-2 expression in the brains of African green monkeys

Abstract

The development of several neurodegenerative disorders, such as Parkinson’s disease, has been linked with decreased mitochondrial performance, leading to oxidative stress as a result of increased production of reactive oxygen species (ROS). Previous studies have established that the mitochondrial enzyme, paraoxonase-2 (PON2), possesses potent antioxidant and anti-inflammatory properties, with its expression linked with lower ROS levels. The aim of this study was to explore the sex-based variations in the protein level of PON2 in different brain regions (striatum, hippocampus, occipital cortex, and dorsolateral prefrontal cortex) of African green monkeys. Our results revealed that the PON2 expression in females was significantly higher than in males, in each of the examined brain regions. As Parkinson’s disease is more prevalent in males compared with females and is characterized by oxidative stress in the nigrostriatal system, these findings add to the growing evidence for PON2 as a target for development of therapeutics to combat this disorder.

Graphical Abstract

1. Introduction

Paraoxonase-2 (PON2) is a 43 kDa protein located in the inner mitochondrial membrane and endoplasmic reticulum which possess potent antioxidant and anti-inflammatory properties [1-3]. PON2 is necessary for properly functioning mitochondria, as PON2-deficient mice display mitochondrial dysfunction [2]. PON2 appears to mitigate oxidative stress by decreasing the production of reactive oxygen species (ROS) possibly by enhancing the function of coenzyme Q associated with complex III of the mitochondrial electron transport chain [1-3]. These attributes of PON2 are strongly supported by evidence that PON2-deficient mice display enhanced mitochondrial oxidative stress, reduced electron transport chain complex I & III activity, oxygen consumption, and ATP levels, whereas PON2 overexpression efficiently protects mitochondria from oxidative stress-induced mitochondrial dysfunction [2-3]. PON2 has been identified in different mouse brain regions, with greatest expression in areas highly innervated by dopamine [4- 6], and it may not be a coincidence that the protective effect of PON2 is pronounced in the dopamine-rich regions such as the striatum because nigrostriatal dopamine neurons are exceptionally susceptible to oxidative stress [7]. Moreover, PON2 deficiency renders neurons more vulnerable to oxidative stress induced by mitochondrial toxin 1-methyl-4-phenylpyridinium [8], the active metabolite derived from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is a potent neurotoxin that causes Parkinsonism in humans and is used experimentally to damage dopamine neurons in animal models of Parkinson’s disease [9]. In addition to an uneven distribution of PON2 among mouse brain regions, there is a difference in PON2 expression between sexes [6, 8]. Specifically, female mice have up to 3-fold higher levels of PON2 than male mice, with the largest differences seen in dopamine-rich regions [5]. Furthermore, data from ovariectomized mice have demonstrated that the higher level of estradiol in females is responsible or contributes to this difference [6, 8]. The greater expression of PON2 in female brains may confer protection against oxidative stress and neuronal degeneration and it is possible that this is relevant to more frequent occurrence of Parkinson’s disease in males [10]. Intriguingly, preclinical studies have confirmed a gradual decline in the expression of PON2 with the age in rodents and nonhuman primates which might contribute to the higher prevalence of oxidative stress and neurodegeneration with aging [1-2]. Recently, PON2 emerged as a new therapeutic target to attenuate oxidative stress, mitochondrial dysfunction, and inflammation associated with neurodegenerative disorders [2-3]. Stimulating PON2 expression in brain might be especially beneficial in protecting male brains from neurodegeneration, if rodent data regarding a sex difference in PON2 expression can be confirmed in primate brain. Nonhuman primates share greater homology to humans than rodents in all respects including genomics, biochemistry, endocrinology, physiology, pharmacology, neuronal network complexity and behavioral specializations [11-13]. Therefore, the present was carried out to determine whether a sex-based disparity in the PON2 protein level exists across different brain areas of nonhuman primates (African green monkeys).

2. Methods

2.1. Non-human primate samples

African green monkeys (Chlorocebus sabaeus) were housed at the St. Kitts Biomedical Research Foundation, an AAALAC accredited facility. All tissue samples were from animals that served as controls for different studies each with their own protocol approved by the Institutional Animal Care and Use Committee. Euthanasia was carried out after injection of an overdose of pentobarbital. Brains were perfused with cold saline, and the following regions dissected from 4 mm sections, placed in cryotubes then frozen in liquid nitrogen: putamen, hippocampus, occipital cortex, dorsolateral prefrontal cortex. Tissue samples were shipped to New Haven, Connecticut in a cryogenic liquid nitrogen vapor shipper and then stored in a freezer set to maintain at −70°C until analysis.

2.2. Total protein measurement

Tissue was sonicated in cold lysis buffer (Cell Signaling Technology, Danvers, MA) with complete™ Protease Inhibitor Cocktail (Roche) and then centrifuged for 15 min at 28,000 × g at 4°C. Total protein content in the supernatant was determined by the BCA assay (Pierce™ BCA protein assay kit, Thermo Scientific, Rockford, IL, USA).

2.3. Western blot analysis

The Western blot protocol was carried out using Bio-Rad (Hercules, CA) equipment and consumables for stain-free protein quantification, following the manufacturer's instructions. Following determination of protein concentration, supernatant was placed in 4x Laemmli loading buffer and protein denatured by placement in a heating block for 5 min at 100 °C. Separation of proteins was achieved on stain-free mini-Protean TGX gels before transfer to nitrocellulose membranes using the turbo transfer method and imaged with the ChemiDoc imaging system. Membranes were then blocked for 1 h at room temperature with 5% nonfat dry milk in Tris buffered saline wash buffer containing 0.1% Tween 20. Following this, membranes were incubated overnight in blocking buffer at 4°C with rabbit monoclonal PON2 antibody (1:1000; ab183710, Abcam, Cambridge, MA). Membranes were washed and then incubated for 2 h at room temperature with anti-rabbit IgG, HRP-linked Antibody (1:10000; #7074, Cell Signaling Technology, USA) in blocking buffer. After washing, the antibody complex was visualized by Clarity chemiluminescence (Bio-Rad Laboratories) and imaged with the ChemiDoc imaging system. PON2 expression was normalized to total lane protein using Image Lab software (Bio-Rad Laboratories) within ChemiDoc XRS+ (Bio-Rad Laboratories, Hercules, CA).

2.3. Statistical analysis

Data are expressed as the mean ± SEM of each group. The normality of the data was assessed by the Shapiro-Wilk tests and by quantile-quantile (QQ) plots. Values from males and females in each region were compared by two-tailed unpaired Student's t-test (α = 0.05), using Prism 8 (GraphPad, La Jolla, CA). p<0.05 was considered statistically significant.

3. Results

Male and female data from each examined brain region passed the Shapiro-Wilk normality test (α = 0.05). For each examined brain region PON2 expression in samples from female monkeys was significantly higher than in males i.e., striatum (2-tailed t (4) = 4.63, p= 0.0094) (Fig. 1B), hippocampus (2-tailed t (4) = 4.7, p= 0.0093) (Fig. 2B), occipital cortex (2-tailed t (4) = 4.75, p= 0.0089) (Fig. 3B) and dorsolateral prefrontal cortex (2-tailed t (4) = 4.9, p= 0.0080) (Fig. 4B). The mean PON2 expression in striatum, hippocampus, occipital cortex and dorsolateral prefrontal cortex brain region was 2.1, 1.7, 1.9 and 1.3-fold higher respectively in female monkeys when compared to male monkeys; the mean difference across these regions is 1.75-fold.

Fig 1:

PON2 protein expression in the striatum region of male and female African green monkeys (n=3). [A] Normal QQ plot of male and female data. [B] PON2 expression in African green monkeys relative to male monkeys. Optical density of PON2 bands were first normalized to total protein per lane and then to male group. [C] Representative blot showing PON2 expression in striatum region, and [D] corresponding image of total protein.

Fig 2:

PON2 protein expression in the hippocampus region of male and female African green monkeys (n=3). [A] Normal QQ plot of male and female data. [B] PON2 expression in African green monkeys relative to male monkeys. Optical density of PON2 bands were first normalized to total protein per lane and then to male group. [C] Representative blot showing PON2 expression in hippocampus region, and [D] corresponding image of total protein.

Fig 3:

PON2 protein expression in the occipital cortex region of male and female African green monkeys (n=3). [A] Normal QQ plot of male and female data. [B] PON2 expression in African green monkeys relative to male monkeys. Optical density of PON2 bands were first normalized to total protein per lane and then to male group. [C] Representative blot showing PON2 expression in occipital cortex region, and [D] corresponding image of total protein.

Fig 4:

PON2 protein expression in the dorsolateral prefrontal cortex of male and female African green monkeys (n=3). [A] Normal QQ plot of male and female data. [B] PON2 expression in African green monkeys relative to male monkeys. Optical density of PON2 bands were first normalized to total protein per lane and then to male group. [C] Representative blot showing PON2 expression in dorsolateral prefrontal cortex region, and [D] corresponding image of total protein.

4. Discussion

The results of present study reveal that in African green monkeys PON2 protein expression in each examined brain region is significantly higher in females than males. These data represent the first characterization of sex-specific PON2 expression in brain of nonhuman primates. Despite nonhuman primates sharing greater homology to humans than rodents, the current result is consistent with findings on PON2 in mice [4-7]. This indicates that the sex bias in brain PON2 expression is an important evolutionary conserved feature, although the basis for the difference and its implications have not been fully resolved. There is widespread recognition that ROS-induced oxidative stress plays a key role in normal brain function as well as the pathology of aging and neurodegenerative disorders like Parkinson’s disease [14]. Particularly relevant to the current data are the well-established findings that female brains exhibit less oxidative stress than males, which may contribute to the longer life span and lower incidence in females of disease such as Parkinson’s disease [15]. There is firm evidence that the actions of the estradiol are largely responsible for the sex differences in redox homeostasis and neuroprotection. However, the exact mechanisms by which estradiol exerts these effects is unclear, although this probably is a consequence of the multifaceted actions of estradiol, including impacts on genomic and non-genomic pathways [15-18]. Biochemical components that regulate oxidative stress are key therapeutic targets for neurodegenerative disorders, and PON2 is a relevant candidate in this context, as it is a novel major intracellular factor that protects brain cells against oxidative stress as well as conferring preferential gender-dependent resistance to oxidative stress [6]. Additionally, in mouse and nonhuman primate brain PON2 protein levels peak early in life and decrease with age [2]. Thus, the lower expression of PON2 in males and in the aging brain may have broad ramifications for susceptibility to age-related neurodegenerative diseases involving oxidative stress, like Parkinson’s disease [2, 19]. PON2 appears to be a critical antioxidant in the CNS and a pertinent mitochondrial enzyme involved in neuroprotection. Our recent finding that pioglitazone activates PON2 expression in-vivo [19] demonstrates that this enzyme is a valid drug target and its pharmacology should be considered further as a potential strategy for neurodegenerative diseases like Parkinson’s disease where oxidative stress plays a significant role in the pathophysiology.

Highlights.

• Paraoxonase-2 possesses potent antioxidant and anti-inflammatory properties and its expression decreases with age.

• Paraoxonase-2 protein expression in female monkeys was significantly higher than in male monkeys in multiple brain regions.

• Reduced Paraoxonase-2 expression may contribute to the higher incidence of some disorders in males.

• Paraoxonase-2 is a therapeutic target for oxidative stress-associated neurodegenerative disorders such as Parkinson’s disease.

Abbreviations:

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

PON2

Paraoxonase-2

ROS

reactive oxygen species