Effect of Premortem and Postmortem Factors on the Distribution and Preservation of Antioxidant Activities in the Cytosol and Synaptosomes of Human Brains

Abstract

The human brain displays oxidant and antioxidant markers with regional specificity that directly impinges on neuronal function in aging and in disease states. Similarly, the antioxidant activities might exhibit differential intracellular distribution rendering subcellular structures differentially vulnerable to toxic insults. To investigate the subcellular distribution of antioxidant activities in the human postmortem brain, we assayed superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in the cytosol and synaptosomal fraction from the frontal cortex (FC) of 45 postmortem human brains. We also tested whether these activities were altered by premortem and postmortem factors, including increasing storage time (11.8–104.1 months), postmortem interval (PMI) (2.5–26 h), age and gender differences, and agonal state [based on Glasgow coma scale (GCS): range: 3–15]. Overall, the antioxidant activities were found to be several folds lower in the synaptosomes compared to cytosol, which could make it more susceptible to degeneration. The activities were significantly affected mainly by age (SOD increased in synaptosomes, p=0.01; GSH decreased in cytosol, p=0.03; GPx decreased in cytosol and increased in synaptosomes, p=0.05; GST decreased in synaptosomes, p=0.05) and to a lesser extent by other premortem (GST decreased with GCS in synaptosomes, p=0.02) and postmortem factors (GSH decreased with PMI in cytosol, p=0.04). Increasing storage time or gender difference did not affect the antioxidant activities. We infer that premortem and postmortem factors in general, and increasing age in particular, significantly alter the antioxidant activities in subcellular fractions of postmortem brain with implications for studies on brain pathology employing stored human samples.

Introduction

There are differences in the status of oxidant and antioxidant markers in different regions of the human brain that might influence the response to aging and neurodegeneration. For instance, regional differences in oxidative damage of the brain in Alzheimer's disease (AD) might occur due to altered antioxidant function and rates of oxygen consumption.^1,2 Two recent studies from our group³ [Venkateshappa et al, unpublished observations) demonstrated that in Parkinson's disease (PD) and AD, age-dependent oxidative stress, mitochondrial damage, and astrogliosis were restricted to the neuroanatomical areas involved in causing disease such as substantia nigra, striatum, and hippocampus, while other regions were unaffected. These data suggest that inherent biochemical differences exist in different brain regions during physiological and pathological conditions that may underlie regional susceptibility. Extrapolating this phenomenon, we questioned whether subcellular compartments in the human brain have varied distribution of oxidant and antioxidant markers. Since these compartments are physically linked, physiological processes occurring in one intracellular compartment might influence the other despite maintaining their identity in terms of membrane structure, expression of distinct proteins and activity of specific channels and receptors. Oxidative stress in the cytosolic⁴ could ultimately lead to synaptic dysfunction and neurodegeneration.^5,6 Similarly, oxidative DNA damage in the nucleus can influence the status of synaptic proteins.^7,8 Likewise, the cytosol GSH pool is crucial for maintaining plasma membrane integrity and ATP level in the synaptosomes.⁹ Synaptic mitochondria display increased sensitivity to oxidative stress.^10,11 Postsynaptic terminals are the initial targets for neurodegeneration due to higher calcium influx and oxidative stress.¹² Therefore, the relative antioxidant activities in synaptosomes and cytosol need to be investigated, which in turn can explain the role of these parameters in neuronal function and degeneration.

Studies on markers of antioxidant function and oxidative stress in human material require use of postmortem brain tissues. Prior to embarking on utilizing postmortem human tissues for study of disease states, it is critical to determine if the preservation and subcellular distribution of antioxidant markers in the human brain could be influenced by premortem parameters (age, gender, medication received, and the agonal state) and postmortem parameters [postmortem interval (PMI), tissue preservation and duration, and the conditions of storage]. The effects of these factors on RNA stability, gene expression,^13–19 and proteins²⁰ in the human brain have been studied, but their influence on antioxidant activities and relative distribution in subcellular compartments have not been elucidated. We have demonstrated that glutathione (GSH) metabolism and markers of oxidative damage and astrogliosis are affected by premortem and postmortem factors.^21,22 Similarly, if the status of antioxidant markers in the subcellular compartments of the human brain is altered independent of disease-related changes, this could influence the interpretation of the experimental results.

We analyzed the status of antioxidant markers in cytosol and synaptosomes of the frontal cortex (FC) in postmortem brains (n=45) stored at the Human Brain Tissue Repository (HBTR), National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India, and determined the effects of increasing PMI (2.5 to 26 h), age (2 days to 80 years), gender, agonal state [based on Glasgow coma scale (GCS): range: 3–15], and prolonged storage time (11.8–104.1 months) at−80°C.

Materials and Methods

All the chemicals used were of analytical grade. Bulk chemicals were obtained from Merck (Whitehouse Station, NJ) and Sisco Research Laboratories Pvt. Ltd. (Mumbai, Maharashtra, India). Fine chemicals and protease inhibitor cocktail were procured from Sigma (Eugene, OR).

Human tissue samples

Human brain samples were sourced from the Human Brain Tissue Repository (HBTR), a National research facility, Department of Neuropathology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India. The brain tissues were collected following written informed consent from close relatives of the deceased to store and utilize the material for research. The Institutional Ethics Committee approval was obtained for the study protocol. The brains were from subjects who succumbed to road traffic accidents (nonalcoholics, nondiabetics, not on any medication, and with no known neurological or psychiatric disorders). Demographic and clinical details of all the subjects and the agonal state scored by the Glasgow coma scale (GCS) were collected from medical records. The GCS score of the subjects was based on (i) best eye response (E; range: 1–4) (ii) best verbal response (V; range: 1–5) and (iii) best motor response (M; range: 1–6) with a total GCS score range of 3 to 15 (noted at the time of hospital admission), with score of 3 corresponding to the most severe agonal state. The hospital stay following admission varied among the patients. During the hospital stay, assisted ventilation and parenteral administration of mannitol, hydrocortisone, and ionotropic drugs were administered to maintain the vital parameters whenever necessary.

Following the death of the patients, the PMI (the time interval between the time of death and placing the dissected brain slices into the freezer maintained at−80 °C) was recorded. Within one hour of death, routinely, the body was transferred to a refrigerator maintained at 2°C–4°C with a recorder and uninterrupted power supply. Following autopsy, the brains were recovered and sliced coronally and kept flat on salt-ice mixture (-15°C to−18°C) during dissection and then transferred in plastic zip lock bags into a box to be stored at−80°C in the HBTR. The procedure of dissection took ∼30–45 min and the brain slices transported on salt-ice mixture were transferred immediately into the deep freezer. The brain areas chosen for the study were anatomically farthest from the site of injury and without distinct edema or grossly apparent pathology. While the major portion of the tissue was frozen for biochemical studies, a portion corresponding to the mirror image of the stored tissue bits were fixed in buffered formalin. These tissues were subjected to histopathological assessment and the samples that maintained tissue integrity were utilized for the study (data not shown). The protocol of autopsy, tissue handling, and other procedures were uniform for all the samples. Such tissues have been earlier utilized extensively as control samples in PD research^23–25 and other studies.^{3,20,21,26–28}

In the current study, frontal cortex (FC) was analyzed from 45 postmortem brains (Table 1). The gender distribution (30 males; 15 females), age (range: 2 days to 80 y), PMI (range: 2.5–26 h), storage time (11–104.1 months), and agonal state (GCS score: 3–15) were noted in all the cases.

Table 1.

List of Human Postmortem Brain Samples Utilized in this Study. The Table Shows All Autopsy Brain Samples and the Premortem and Postmortem Factors of the Tissues (Age, Gender, PMI, Agonal State and Storage Period)

Sample No.	Gender	Age (y)	PMI (h)	Status of the patient	GCS rating (at the time of hospital admission)	Storage (months)
1	F	0.005	22.0	All samples utilized in the current study were obtained from subjects who succumbed to head injury (neurotrauma) and the severity of the injury is indicated by the GCS score (See methods)	12	39.5
2	F	1	25.3		4	91.3
3	F	2	26.0		NA	104.1
4	M	2	18.3		6	93.7
5	M	2	10.0		14	38.6
6	M	4	5.5		3	99.2
7	F	12	5.5		7	44.4
8	M	13	15.0		NA	85.6
9	M	14	3.5		8	38.5
10	M	15	24.5		8	88.5
11	F	15	10.5		15	38.0
12	M	16	7.0		NA	36.7
13	M	17	12.0		5	20.0
14	M	23	6.3		3	14.6
15	M	24	13.0		7	73.2
16	M	24	7.5		5	20.4
17	F	25	12.0		3	41.6
18	M	25	7.5		3	35.3
19	M	25	14.5		15	35.1
20	M	27	6.5		NA	64.6
21	F	27	4.0		11	34.7
22	F	29	18.5		10	81.7
23	M	35	6.3		4	67.2
24	M	35	7.5		11	39.8
25	M	40	10.0		14	75.2
26	F	40	16.0		4	40.1
27	M	40	7.0		3	39.9
28	M	40	19.0		9	17.4
29	M	45	2.5		13	41.2
30	F	45	7.5		3	40.8
31	F	45	17.8		3	20.5
32	M	48	3.0		3	41.7
33	M	50	15.0		4	40.6
34	M	50	19.3		5	11.8
35	M	51	12.0		NA	81.1
36	M	55	7.0		15	74.6
37	M	60	20.0		4	85.3
38	M	65	22.0		6	92.4
39	M	65	13.2		3	84.1
40	M	70	19.5		NA	103.2
41	F	70	9.0		6	16.5
42	F	77	20.5		3	13.6
43	F	80	3.1		3	16.2
44	M	80	15.8		4	16.2
45	F	80	6.0		3	16.2

PMI, postmortem interval; GCS, Glasgow Coma Scale; NA, Not available.

Isolation of cytosol and synaptosomes from brain tissue²⁹

Approximately 200 mg of brain tissue was minced in 1000 μl of isolation buffer (350 mM sucrose, 5 mM TES, and 1 mM EGTA, pH 7.2) containing 100 μl of protease inhibitor cocktail and manually homogenized (16 strokes). The homogenate was centrifuged at 1000 g for 5 min at 4°C, and the supernatant was stored. The pellet was homogenized again (16 strokes) on ice and centrifuged (1000 g, 5 min at 4°C). The supernatants from both the steps were pooled and centrifuged at 8500 g for 10 min at 4°C to obtain the crude mitochondrial/synaptosome pellet. The post-mitochondrial supernatant that corresponded to the cytosol fraction was aliquoted and stored. The pellet was re-suspended in 300 μl of isolation buffer and overlaid on a discontinuous Ficoll gradient consisting of 6% (w/v Ficoll, ρ=1.065 g/ml, 3 ml), 9% (w/v Ficoll, ρ=1.075 g/ml, 2 ml), and 12% (w/v Ficoll, ρ=1.085 g/ml, 3 ml). The gradient was centrifuged at 75,000 g for 1 h at 4°C, and the synaptosomes which formed a whitish ring in the middle of the gradient were collected, resuspended in reconstitution buffer (250 mM sucrose and 10 mM TES, pH 7.2) and stored as aliquots in−80 °C. The purity and integrity of the isolated synaptosomes were confirmed by electron microscopy (TEM-Tecnai G2) (Fig. 1) as described previously (30). The cytosol and synaptosomes were utilized for the following biochemical experiments after protein estimation (31).

FIG. 1.

Representative electron micrograph showing synaptosomes (magnification=1X 13,000) purified from postmortem human brains samples (frontal cortex).

Superoxide dismutase (SOD) assay

SOD activity was assayed using its inhibitory action on quercetin oxidation based on the method described earlier,³² with minor modifications. The final reaction mixture contained 30 mM Tris HCl (pH 9.1), 0.5 mM EDTA, 50 mM TEMED, 0.05 mM quercetin, and 10 μl of mitochondria containing 10 μg of protein. The reaction was monitored at 406 nm for 10 min. One unit of SOD activity was defined as the amount of enzyme (per mg protein) that inhibits quercetin oxidation reaction by 50% of maximal value.

Estimation of total glutathione (GSH+GSSG)

Total glutathione estimations in the brain extracts were carried out by the DTNB recycling method, as described earlier.³³ All estimations were conducted in triplicate, and total glutathione concentrations were normalized per mg protein.

Glutathione-S-transferase (GST) assay

GST was assayed by the 1-chloro 2-4-dinitro benzene (CDNB) method.³⁴ To 1 ml reaction mixture containing phosphate buffer (0.1 M, pH 6.5; 0.5 mM EDTA), CDNB (1.5 mM), and 50 μl GSH (1 mM), 30 μg protein (sample) were added and the increase in absorbance at 340 nm was monitored for 5 min. The enzyme activity was expressed as nmoles of S-2,4, dinitrophenyl glutathione formed /min/mg protein (MEC=9.6 mM-1cm−1).

Glutathione peroxidase (GPx) assay

GPx activity was determined by t-butyl hydroperoxide (tbHP) method.³⁵ The reaction mixture containing 150 μg protein (sample), phosphate buffer (0.1 M), 0.5 mM EDTA, 100 μl glutathione reductase (0.24 U), 100 μl GSH (1 mM), and 100 μl NADPH (0.15 mM) was incubated at 37°C for 3 min and the reaction was initiated by the addition of 100 μl tbHP (0.12 mM). Change in absorbance at 340 nm was monitored for 5 min spectrophotometrically and the activity was expressed as nmoles of NADPH oxidized /min/mg protein (MEC=6.22 mM⁻¹cm ⁻¹).

Glutathione reductase (GR) assay

Solubilized brain protein extracts (100 μg) was assayed at 25°C in 0.1 M Tris–HCl (pH 8.1) and 0.2 mM NADPH, and the reaction was initiated by the addition of 1 mM GSSG and followed spectrophotometrically at 340 nm.³⁶

Statistical analysis

Quantitative data were accumulated from at least three independent experiments and expressed as mean±SD, followed by analysis of variance (ANOVA) and Pearson's correlation of linear regression (r value). In all the experiments, data with p<0.05 were considered to be statistically significant.

Results

In the current study, we investigated the distribution of antioxidant activities in the subcellular fractions of postmortem human brains and tested whether they are influenced by increasing storage time at−80°C (11.8–104.1 months), PMI (range: 2.5–26 h), age of the donors (2 days to 80 y), gender of the subject, and agonal state (GCS score: 3–15). This was carried out by assaying the SOD, GSH, GPx, GR, and GST in cytosol and synaptosomes fractions of FC from human brain samples (n=45).

The SOD activity was>5 fold lower in synaptosomes compared to the cytosol. The SOD activity was unaffected by storage time (cytosol: r=− 0.12, p=0.2; synaptosomes: r=− 0.10, p=0.29), PMI (cytosol: r=0.02, p=0.46; synaptosomes: r=− 0.11, p=0.24), agonal state (cytosol: r=− 0.12, p=0.23; synaptosomes: r=0.04, p=0.40), or gender (cytosol: p=0.89; synaptosomes: p=0.37) (Fig. 2). However, SOD activity increased with age in the synaptosomes, while it remained relatively unaltered in the cytosol fraction (cytosol: r=0.20, p=0.10; synaptosomes: r=0.37, p=0.01) (Fig. 2D).

FIG. 2.

Effect of premortem and postmortem factors on superoxide dismutase (SOD) activity in cytosol and synaptosomes of the frontal cortex (FC) region of human postmortem brains (n=45). SOD activity in cytosolic and synaptosomes from FC was assayed in triplicate, and the average for each sample was plotted as mean±SD followed by regression analysis. The r and p values are shown. Scatter plot graph with linear regression line for SOD levels with increasing (A) storage time, (B) PMI, (C) agonal state, (D) age, and with (E) gender are shown.

The total GSH content was ∼5 fold lower in the synaptosomes compared to cytosol. The total GSH content was unaffected by storage time (cytosol: r=0.21, p=0.11; synaptosomes: r=− 0.11, p=0.27), agonal state (cytosol: r=− 0.12, p=0.24; synaptosomes: r=− 0.23, p=0.09), or gender (cytosol: p=0.35; synaptosomes: p=0.15) (Fig. 3). However, total GSH decreased in the cytosol with increasing PMI (cytosol: r=− 0.28, p=0.04; synaptosomes: r=0.05, p=0.38) (Fig. 3B) and age, while it was unaltered in synaptosomes (cytosol: r=− 0.28, p=0.03; synaptosomes: r=0.11, p=0.24) (Fig. 3D).

FIG. 3.

Effect of premortem and postmortem factors on the total glutathione (GSH) content in cytosol and synaptosomes of the frontal cortex (FC) region of human postmortem brains (n=45). Total GSH content in cytosol and synaptosomes from FC was assayed in triplicate, and the average for each sample was plotted as mean±SD followed by regression analysis. The r and p values are shown. Scatter plot graph with linear regression line for GSH levels with increasing (A) storage time, (B) PMI, (C) agonal state, (D) age, and with (E) gender are shown.

On the other hand, GPx activity was ∼ 8 fold lower in synaptosomes compared to cytosol. GPx activity was unaffected by storage time (cytosol: r=0.05, p=0.38; synaptosomes: r=− 0.15, p=0.18), PMI (cytosol: r=− 0.11, p=0.25; synaptosomes: r=− 0.11, p=0.24), agonal state (cytosol: r=0.13, p=0.23; synaptosomes: r=− 0.13, p=0.22) or gender (cytosol: p=0.93; synaptosomes: p=0.23) (Fig. 4). Interestingly, while the GPx activity decreased with age in cytosol, the activity significantly increased in synaptosomes (cytosol: r=− 0.26, p=0.05; synaptosomes: r=0.25, p=0.05) (Fig. 4D).

FIG. 4.

Effect of premortem and postmortem factors on glutathione peroxidase (GPx) activity in cytosol and synaptosomes of the frontal cortex (FC) region of human postmortem brains (n=45). GPx activity in cytosol and synaptosomes from FC was assayed in triplicate, and the average for each sample was plotted as mean±SD followed by regression analysis. The r and p values are shown. Scatter plot graph with linear regression line for GPx levels with increasing (A) storage time, (B) PMI, (C) agonal state, (D) age, and with (E) gender are shown.

The GR activity was ∼ 20 % lower in synaptosomes compared to cytosol. GR activity was unaffected by all factors including storage time (cytosol: r=0.11, p=0.27; synaptosomes: r=− 0.18, p=0.14), PMI (cytosol: r=− 0.13, p=0.20; synaptosomes: r=− 0.05, p=0.36), agonal state (cytosol: r=0.13, p=0.23; synaptosomes: r=0.19, p=0.13), age (cytosol: r=− 0.10, p=0.26; synaptosomes: r=− 0.08, p=0.30) and gender (cytosol: p=0.57; synaptosomes: p=0.11) (Fig. 5).

FIG. 5.

Effect of premortem and postmortem factors on glutathione reductase (GR) activity in cytosol and synaptosomes of the frontal cortex (FC) region of human postmortem brains (n=45). GR activity in cytosol and synaptosomes from FC was assayed in triplicate, and the average for each sample was plotted as mean±SD followed by regression analysis. The r and p values are shown. Scatter plot graph with linear regression line for GR levels with increasing (A) storage time, (B) PMI, (C) agonal state, (D) age, and with (E) gender are shown.

The total GST activity was>20 fold lower in synaptosomes compared to cytosol. The GST activity was unaffected by storage time (cytosol: r=0.07, p=0.34; synaptosomes: r=− 0.18, p=0.15), PMI (cytosol: r=− 0.01, p=0.47; synaptosomes: r=0.07, p=0.33), or gender (cytosol: p=0.72; synaptosomes: p=0.91) (Fig. 6). However, GST activity decreased in synaptosomes with agonal state (cytosol: r=0.12, p=0.23; synaptosomes: r=− 0.27, p=0.05) (Fig. 5C) and age, while it was unaltered in cytosol (cytosol: r=0.05, p=0.38; synaptosomes: r=0.32, p=0.02) (Fig. 6D).

FIG. 6.

Effect of premortem and postmortem factors on glutathione-S-transferase (GST) activity in cytosol and synaptosomes of the frontal cortex (FC) region of human postmortem brains (n=45). GST activity in cytosol and synaptosomes from FC was assayed in triplicate, and the average for each sample was plotted as mean±SD followed by regression analysis. The r and p values are shown. Scatter plot graph with linear regression line for GST levels with increasing (A) storage time, (B) PMI, (C) agonal state, (D) age, and with (E) gender are shown.

Overall, we observed that the antioxidant activities were several fold lower in synaptosomes as compared to cytosol in the postmortem human brains. The activities were affected mainly by the age and to a lesser extent by other premortem and postmortem factors (Table 2).These results should be considered during interpretation of biochemical data in a meaningful manner.

Table 2.

Comparison of Antioxidant Activities in the Total Extracts (Total) and Subcellular Compartments (Fractions) of the Frontal Cortex from Postmortem Human Brains ( n =45). The Table Indicates That While Gender Does Not Affect Any of the Parameters, Age of the Samples Significantly Affects the Activities in Different Fractions

		Results
		Storage time (11.8–104.1 mo)		PMI (2.5–26 h)		Age (2d–80 y)		GCS (15–3:increasing severity)		Gender
Sl. No.	Assay	Total ^@	fractions	Total ^@	fractions	Total ^&	Fractions	Total ^@	fractions	Total ^@	Fractions
1.	SOD	Ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns
			c^$=ns		c^$=ns		c^$=ns		c^$=ns		c^$=ns
			0s^$=ns		s^$=ns		s^$: r=0.37		s^$=ns		s^$=ns
							p=0.01
2.	Total GSH	Ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns
			c^$=ns		c^$: r=−0.28		c^$: r=−0.28		c^$=ns		c^$=ns
					p=0.04		p=0.03
			s^$=ns		s^$=ns		s^$=ns		s^$=ns		s^$=ns
3.	GPx	Ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#=ns
			c^$=ns		c^$=ns		c^$:r=−0.26		c^$=ns		c^$=ns
							p=0.05
			s^$=ns		s^$=ns		s^$:r=0.25		s^$=ns		s^$=ns
							p=0.05
4.	GR	Ns	m^#=ns	ns	m^#=ns	ns	m^#=ns	ns	m^#: r=−0.41	ns	m^#=ns
									p=0.003
			c^$=ns		c^$=ns		c^$=ns		c^$=ns		c^$=ns
			s^$=ns		s^$=ns		s^$=ns		s^$=ns		s^$=ns
5.	GST	Ns	m^#:r=− 0.39	ns	m^#=ns	r=0.43	m^#=ns	ns	m^#=ns	ns	m^#=ns
			p=0.005			p=0.03
			c^$=ns		c^$=ns		c^$=ns		c^$=ns		c^$=ns
			s^$=ns		s^$=ns		s^$: r=0.32		s^$: r=−0.27		s^$=ns
							p=0.02		p=0.05

m, mitochondria; c, cytosol (cyt); s, synaptosome (syn); SOD, superoxide dismutase; GSH, glutathione; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-s-transferase; PMI, postmortem interval; GCS, Glasgow coma scale; ns, not significant.

Previous study (22); ^#Recent study (Harish et al, 2011, manuscript submitted); ^$Current study; ^&Unpublished observations (Venkateshappa et al.).

The r and p values for the parameters with significant change are indicated in boldface.

Discussion

The differential distribution of antioxidant markers in subcellular compartments must be studied to understand its influence on brain function and pathology. Among these markers, intracellular distribution of total GSH has been studied extensively in animal models. Schnellmann et al.³⁷ examined the intracellular distribution of total GSH in rabbit renal tubules and found that irrespective of the in vivo GSH levels in the tubules, the mitochondrial GSH pool was unaltered. Although the mitochondrial GSH concentration accounted for ∼10% of the total cellular GSH, depletion of total GSH in the tubules caused selective depletion of the cytosolic pool without affecting the mitochondrial GSH pool. Further,>40% loss of mitochondrial GSH caused mitochondrial dysfunction, reflecting complicated GSH dynamics between cytosol and mitochondria and their role in mitochondrial function. In a related study, Roychowdhury et al.³⁸ studied whether the mitochondrial and cytosolic GSH are differentially affected by endogenous nitric oxide (NO) in microglial cultures. Activated inducible nitric oxide synthase (iNOS) significantly depleted total GSH but the mitochondrial GSH was relatively unaffected. NO-mediated oxidative damage and mitochondrial dysfunction were more pronounced in cells where mitochondrial GSH was depleted compared to cytosolic GSH. Further, endogenous NO production did not compromise the mitochondrial GSH pool which, in turn, might explain the ability of these cells to resist the damaging effects of high-output NO production. Previously, we demonstrated that total GSH content in whole tissue extracts of FC from postmortem human brains was unaffected by premortem and postmortem factors. Similarly, total GSH in mitochondria (Harish et al, manuscript submitted) and synaptosomes (current study) in FC from human brains was unaffected by premortem and postmortem factors. In contrast, cytosolic GSH was significantly decreased with increasing age and PMI (Fig. 3). This is consistent with a previous study⁹ which investigated the effect of GSH depletion on the viability of synaptosomes during brain aging in mice. Although aging did not influence the GSH and ATP levels and the viability of mouse synaptosomes, depletion of cytosolic GSH decreased the ATP levels and viability of synaptosomes in aged mice but not in young animals. These results emphasize the importance of the cytosol GSH for the maintenance of the membrane integrity of synaptosomes during brain aging in mice. The antioxidant activities in cytosol and synaptosomes were mainly affected by age and to a lesser extent by other premortem and postmortem factors. Gender had the least effect on antioxidant activities tested (Table 2). On the other hand, other antioxidant activities such as SOD, GPx, and GST were altered in the subcellular fractions with increasing age. We observed opposite trends in the GPx activity with increasing age, with declining activity in cytosol probably compensated by an increase in synaptosomes (Fig. 4D).

The current study also observed several fold lower antioxidant activity in synaptosomes compared to cytosol. All activities were≥5 fold higher in the cytosol compared to synaptosomes, while GR activity was ∼20% increased in cytosol. This also indicates the selective vulnerability of synaptosomes to oxidative damage with implications for pathogenesis of degenerative pathology. This was explained by a recent study,³⁹ which quantitated the distribution of oxidant and antioxidant markers in various subcellular (neuropil) fractions of FC from control, mild cognitive impairment (MCI), moderate AD, and advanced AD human brains. All the neuropil fractions (cytosol, mitochondria, and synaptosomes) demonstrated pathology-dependent elevation in oxidant markers and depletion of antioxidant activities, but the highest oxidative damage was observed in synaptosomes. This might be considered as the mechanistic basis underlying the selective targeting of synaptosomes during neurodegeneration.

Table 2 shows the distribution of antioxidant activities in whole brain extracts and different subcellular fractions in FC of postmortem human brains. Although the antioxidant activities in the whole extracts of FC remained unaffected by premortem and postmortem factors, the subcellular fractions revealed significant alterations. Hence the data from whole cell or tissue extracts should be used cautiously, as it may not accurately reflect the changes in subcellular structures. For instance, while the SOD activity was unaltered with increasing age in total extracts of FC, the activity increased with age in synaptosomes (Fig. 2D). Similarly, the GSH level which was unaltered in the total extracts of FC with increasing age and PMI was significantly decreased in the cytosolic fraction from the same samples indicating that this decrease was masked due to the other fractions (Figs. 3B and 3D). GPx activity in cytosol and synaptosomes was altered with age, while this was not reflected in the whole extracts (Fig. 4D). Similarly, the GST activity which was significantly increased in the whole extracts with increasing age was due to elevation only in the synaptosomes compared to the cytosolic and mitochondrial fractions (Fig. 6D and Table 2). In some instances, the activity in total extract could be correlated with the opposing trends in subcellular fractions. For example, GPx activity revealed decrease in cytosolic and increase in synaptosomal fraction with age, while total extracts showed the activity to be unaltered (Fig. 4D).

Interestingly, gender of the subjects did not influence any of the activities tested in the current study. In our previous study (Harish et al 2011, manuscript submitted), we observed that the activities of mitochondrial enzymes, such as citrate synthase and succinate dehydrogenase, were significantly higher in brains of female subjects compared to males. Gender of the subject might have significant effects on the neuronal function and synaptic transmission and this might be induced due to the action of hormones such as estrogen in the brain. There are different studies that have demonstrated that exposure to estrogen greatly influences the synaptic functions in the brain. For example, estrogen increases the concentration of neurotransmitters such as dopamine, serotonin, and norepinephrine, and modulates their release, uptake, and enzymatic inactivation.⁴⁰ Estrogen is also known to increase the receptors of these neurotransmitters.⁴¹ Estrogens can modulate synaptic functions by inhibiting Na-dependent of Ca²⁺ efflux from the mitochondria. This causes mitochondrial retention of Ca²⁺, which modulates the synaptosomal Ca²⁺ content, thereby affecting the ion homeostasis in nerve terminals.⁴² Further, estrogens have been described to induce synaptogenesis in the principal neurons of hippocampus and are shown to be synthesized and released by these neurons.⁴³

In conclusion, the current study combined with our earlier work²² [Harish et al, manuscript submitted, and Venkateshappa et al, unpublished observations)], reveal a complex distribution of antioxidant activities in FC of postmortem human brain samples which could also differ in various anatomical areas. Further, these activities could be influenced by some premortem and postmortem factors, and the effect could be different in different subcellular fractions. Compared to cytosol, the synaptosomes showed several fold decrease in antioxidant activity which might make it vulnerable to oxidative stress and contribute to evolution of neurodegenerative diseases.

Footnotes

Acknowledgments

The authors gratefully acknowledge the Human Brain Tissue Repository (HBTR), Department of Neuropathology, NIMHANS, Bangalore, India for providing the human brain tissues for the study. The authors gratefully acknowledge Dr. N. Gayathri and Mrs. Hemavathy from the Electron Microscopy Laboratory, Department of Neuropathology, NIMHANS, for assistance in electron microscopy. The authors thank the donors and their relatives for the kind gift of human brains for neurobiological studies.

Disclosure Statement

The authors declare that there are no conflicts of interest.

This study was financially supported by the Indian Council of Medical Research (ICMR IRIS ID No. 2009-07710) (to MMSB). GH is supported by a senior research fellowship from ICMR, India. VC gratefully acknowledges the financial support from Siddhartha Medical College, Tumkur, India.

References

Hensley

, Hall

, Subramaniam

et al. Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation. J Neurochem, 1995; 65:2146–2156.

Wang

, Xiong

, Xie

et al. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease. J Neurochem, 2005; 93:953–962.

Venkateshappa

, Harish

, Mythri

et al. Increased oxidative damage and decreased antioxidant function in aging human substantia nigra compared to striatum: Implications for Parkinson's disease. Neurochem Res, 2012; 37:358–369.

Cash

, Perry

, Ogawa

et al.

Is Alzheimer's disease a mitochondrial disorder?

Neuroscientist, 2002; 8:489–496.

Onyango

, Khan

, Miller

et al. Mitochondrial genomic contribution to mitochondrial dysfunction in Alzheimer's disease. J Alzheimers Dis, 2006; 9:183–193.

Reddy

, Beal

. Are mitochondria critical in the pathogenesis of Alzheimer's disease? Brain Res Brain Res Rev, 2005; 49:618–632.

Forero

, Casadesus

, Perry

et al. Synaptic dysfunction and oxidative stress in Alzheimer's disease: Emerging mechanisms. J Cell Mol Med, 2006; 10:796–805.

Urano

, Asai

, Makabe

et al. Oxidative injury of synapse and alteration of antioxidative defense systems in rats, and its prevention by vitamin. E Eur J Biochem, 1997; 245:64–70.

Martinez

, Ferrandiz

, Diez

et al. Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice. Mech Ageing Dev, 1995; 84:77–81.

10.

Brown

, Sullivan

, Geddes

. Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria. J Biol Chem, 2006; 281:11658–11668.

11.

Naga

, Sullivan

, Geddes

. High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J Neurosci, 2007; 27:7469–7475.

12.

Mattson

, Partin

, Begley

. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res, 1998; 807:167–176.

13.

Burke

, O'Malley

, Chung

et al. Effect of pre- and postmortem variables on specific mRNA levels in human brain. Brain Res Mol Brain Res, 1991; 11:37–41.

14.

Barton

, Pearso

, Najlerahim

et al. Pre- and postmortem influences on brain RNA. J Neurochem, 1993; 61:1–11.

15.

Harrison

, Heath

, Eastwood

et al. The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: Selective mRNA vulnerability and comparison with their encoded proteins. Neurosci Lett, 1995; 200:151–154.

16.

Preece

, Cairns

. Quantifying mRNA in postmortem human brain: Influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance. Brain Res Mol Brain Res, 2003; 118:60–71.

17.

Tomita

, Vawter

, Walsh

et al. Effect of agonal and postmortem factors on gene expression profile: Quality control in microarray analyses of postmortem human brain. Biol Psychiatry, 2004; 55:346–352.

18.

Ervin

, Heinzen

, Cronin

et al. Postmortem delay has minimal effect on brain RNA integrity. J Neuropathol Exp Neurol, 2007; 66:1093–1099.

19.

Durrenberger

, Fernando

, Kashefi

et al. Effects of antemortem and postmortem variables on human brain mRNA quality: A BrainNet Europe study. J Neuropathol Exp Neurol, 2010; 69:70–81.

20.

Chandana

, Mythri

, Mahadevan

et al. Biochemical analysis of protein stability in human brain collected at different post-mortem intervals. Indian J Med Res, 2009; 129:189–199.

21.

Harish

, Venkateshappa

, Mahadevan

et al. Effect of storage time, postmortem interval, agonal state, and gender on the postmortem preservation of glial fibrillary acidic protein and oxidatively damaged proteins in human brains. Biopreserv Biobank, 2011; 9:379–387.

22.

Harish

, Venkateshappa

, Mahadevan

et al. Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains. Neurochem Int, 2011; 59:1029–1042.

23.

Karunakaran

, Saeed

, Mishra

et al. Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. J Neurosci, 2008; 28:12500–12509.

24.

Alladi

, Mahadevan

, Yasha

et al. Absence of age-related changes in nigral dopaminergic neurons of Asian Indians: Relevance to lower incidence of Parkinson's disease. Neuroscience, 2009; 159:236–245.

25.

Mythri

, Venkateshappa

, Harish

et al. Evaluation of markers of oxidative stress, antioxidant function and astrocytic proliferation in the striatum and frontal cortex of Parkinson's disease brains. Neurochem Res, 2011; 36:1452–1463.

26.

Agarwal

, Kommaddi

, Valli

et al. Drug metabolism in human brain: High levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite. PLoS One, 2008; 3:e2337.

27.

Alladi

, Mahadevan

, Shankar

et al. Expression of GDNF receptors GFRalpha1 and RET is preserved in substantia nigra pars compacta of aging Asian Indians. J Chem Neuroanat, 2010; 40:43–52.

28.

Alladi

, Mahadevan

, Vijayalakshmi

et al. Ageing enhances alpha-synuclein, ubiquitin and endoplasmic reticular stress protein expression in the nigral neurons of Asian Indians. Neurochem Int, 2010; 57:530–539.

29.

Douglas

. Rapid, quantitative isolation of mitochondria from rat liver using Ficoll gradients in vertical rotors. Anal Biochem, 1983; 131:453–457.

30.

Frasca

, Parks

. A Routine technique for double-staining ultrathin sections using uranyl and lead salts. J Cell Biol, 1965; 25:157–161.

31.

Bradford

. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976; 72:248–254.

32.

Bagnyukova

, Storey

, Lushchak

. Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation. J Therm Biol, 2003; 28:21–28.

33.

Mythri

, Jagatha

, Pradhan

et al.

Mitochondrial complex I inhibition in Parkinson's disease: How can curcumin protect mitochondria?

Antioxid Redox Signal, 2007; 9:399–408.

34.

Guthenberg

, Alin

, Mannervik

. Glutathione transferase from rat testis. Methods Enzymol, 1985; 113:507–510.

35.

Flohe

, Gunzler

. Assays of glutathione peroxidase. Methods Enzymol, 1984; 105:114–121.

36.

Rigobello

, Folda

, Baldoin

et al. Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase. Free Radic Res, 2005; 39:687–695.

37.

Schnellmann

, Gilchrist

, Mandel

. Intracellular distribution and depletion of glutathione in rabbit renal proximal tubules. Kidney Int, 1988; 34:229–233.

38.

Roychowdhury

, Wolf

, Keilhoff

et al. Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress. Nitric Oxide, 2003; 8:39–47.

39.

Ansari

, Scheff

. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol, 2010; 69:155–167.

40.

JSM

. Estrogen and mood change via CNS activity. Menopausal Med, 1999; 7:4–8.

41.

McEwen

, Alves

. Estrogen actions in the central nervous system. Endocr Rev, 1999; 20:279–307.

42.

Horvat

, Petrovic

, Nedeljkovic

et al. Estradiol affect Na-dependent Ca2+efflux from synaptosomal mitochondria. Gen Physiol Biophys, 2000; 9:59–71.

43.

Kretz

, Fester

, Wehrenberg

et al. Hippocampal synapses depend on hippocampal estrogen synthesis. J Neurosci, 2004; 24:5913–5921.

Effect of Premortem and Postmortem Factors on the Distribution and Preservation of Antioxidant Activities in the Cytosol and Synaptosomes of Human Brains

Abstract

Introduction

Materials and Methods

Human tissue samples

Isolation of cytosol and synaptosomes from brain tissue 29

Superoxide dismutase (SOD) assay

Estimation of total glutathione (GSH+GSSG)

Glutathione-S-transferase (GST) assay

Glutathione peroxidase (GPx) assay

Glutathione reductase (GR) assay

Statistical analysis

Results

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Isolation of cytosol and synaptosomes from brain tissue²⁹