Ca2+ induced lipid peroxidation accentuates aging in rat brain mitochondria

Aging has been ascribed to changes at molecular intersections and impairments due to free radical generation (Harman, 1956), alterations in immunity (Hong et al. , 2019; Martínez de Toda et al. , 2019), telomere shortening (López-Otín et al. , 2013) and the presence of genes that play a vital role in senescence (Payne & Chinnery, 2015). More recently, however, a single amalgamated theory that emphasizes gene repair and maintenance systems, and their surrounding is becoming more and more accepted (Rattan, 2006), stressing upon the need for a holistic, cumulative and logical analysis of senescence. However, massive research integration on aging and aging-related processes should be able to address it systematically and simplify theories on aging. More so, as commonly found by many researchers around the globe, one mechanism that is thought to contribute to this age-associated degeneration is the production of free radicals (Cui et al. , 2012; Sanz, 2016; Stepien et al. , 2017). Reactive oxygen species (ROS) are generated at multiple compartments within the cell and as accidental byproducts of multiple enzymes. Significant contributions include proteins that are localized on the plasma membrane, such as a family of oxidases, as well as the activity of various cytosolic enzymes such as cyclo-oxygenase. Peroxisomal lipid metabolism results in ROS release too. Although all these sources add up to the overall oxidative load, mitochondria still occupy the central position producing approximately 90% of total cellular ROS.

An increase in free radical generation usually worsens the mitochondrial physiology, partly via lipid peroxidation, respiratory chain protein oxidation, and mitochondrial DNA damage. Additionally, damaged mitochondrial membranes would shift the cellular metabolism towards glycolysis signifying the tight interplay between bioenergetics, metabolism and aging (Silva et al. , 2013).

MATERIALS AND METHODS

MATERIALS

Ethylene glycol-bis-(-aminoethyl ether) N, N, N1, N1 tetraacetic acid (EGTA), calcium chloride, sodium succinate, N-2-hydroxy ethyl piperazine-n-2-ethane sulfonic acid (HEPES), and dimethyl sulfoxide and epinephrine were obtained from Sigma Chemicals. Other chemicals of analytical grade were obtained from BDH Industries Ltd. and Glaxo Laboratories, Mumbai, India.

Sprague Dawley albino rats were procured from Sri Raghavendra Enterprises, Bangalore, and acclimatized to laboratory conditions (12 hrs dark/light, 28±2oC). Animals were let free to feed on standard food (Amruth Feeds, India) and potable water ad libitum. Animals were maintained following the stipulated guidelines of ICMR - National Institute of Nutrition, Hyderabad, with the approval taken from Animal Ethical Committee, Bangalore University, Bangalore. Rats of three different age groups: 2-3 weeks, 2-3 months and 2-3 years, were chosen to correlate neonatal, young adult and senescent/old stage of the animal. They were acclimatized a week before the commencement of the study.

METHODS

Isolation of Mitochondria:

1,680g for 10 minutes. Mitochondria collected are stored at -700C till further use. All the isolation procedures were carried out at 0 - 40C.

The protein concentration was determined by the Folin-Lowry method. The total protein concentration made up to 1mg/ ml in storage buffer.

Mitochondrial marker assay

Lipid Peroxidation assay

Briefly, 100ug of mitochondria is treated with varying concentration of Ca2+ (0 to 300uM) along with 2ml of TBA: TCA: HCL reagent in 1:1:1 ratio of 0.37% Thiobarbituric acid, 15% Trichloro-acetic acid and 0.25N Hydrochloric acid; placed on a water bath for 10 minutes, later cooled down and spun at 1000 rpm for another 10 minutes. An aliquot of the solution measured at 535 nm with an extinction coefficient of MDA being 1.56x105 m-1/cm-1, was used for calculating LPO activity. LPO activity is measured with and without Ca2+ to delineate the role of Ca2+ in the ageing brain. The values were expressed in nmoles of MDA/ g wet weight of tissue.

RESULTS

Figure 1 . Effect of Ca²⁺ on LPO activity of mitochondria isolated from brain tissue of three different age groups viz., neonatal, young adult and aged rats. Mitochondria (100 µg/ml protein) isolated from neonatal rats showed low basal rate of LPO activity in control studies and gradient increase in activity with each treatment of rise in Ca²⁺ concentration. Data points represent the Mean ± SD of 5 experiments (p<0.001).

Figure 2. Normal probability plot, illustrating the linear and normal frequency distribution of LPO products formed values to Ca²⁺ treatment in all three age groups. The table represents the results of Two-Way Analysis of Variance with a significant p=0.01.

DISCUSSION

In our extended study, including the role of Ca2+ points out at the gentle rise in rate of LPO indicating the increased ROS production. The LPO rate, though dismal, is quantitative enough to measure the deleterious effects of ROS, suggesting a direct relationship between ROS and LPO. Lipid peroxidation products are highly reactive and include hydroxyl acids or reactive aldehydes (Gaschler & Stockwell, 2017). Assaying the LPO reaction results in intermediate products like PUFA-derived stable aldehydes such as 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA) and acrolein (Ayala et al , 2014). Our experiment measuring malondialdehyde indicate the formation of covalent adducts with lipids (Lykkesfeldt, 2007), DNA (Kadiiska et al , 2005), matrix proteins and inner membrane bound proteins as well (Pizzimenti et al , 2013). Hence, LPO involved in loss of membrane bound proteins resulting in loss of structural integrity of the membrane (Ademowo et al , 2017;

CONCLUSION

We studied the effect of Ca²⁺ on LPO induction in mitochondria isolated from brain tissue of all three age groups viz., neonatal, young adult and aged rats. Aged rats exhibiting an average of ~12% rise in lipid peroxidation to 100 µM Ca²⁺ is indicative of increased sensitivity to Ca²⁺ interplay in LPO reaction. Our results are in agreement with Ca²⁺ actively potentiating LPO (Jain & Shohet, 1981). An application of 300 µM Ca²⁺ further doubled the rate of lipid peroxidation in aged rats to 20%, which should be significantly altering the phospholipid content in the mitochondrial inner membrane, compromising the membrane stability and architecture. In summary, age related free radical production and accumulation has been suggested to be harmful to major components of the cell and would promulgate aging and age-related diseases like Alzheimer’s disease (Simoncini et al. , 2015). The decrease in LPO activity as seen in young adult rats may be due to stable and healthy adult life, as seen in all animals.

ACKNOWLEDGMENT