Elsevier

Process Biochemistry

Volume 84, September 2019, Pages 180-185
Process Biochemistry

Attenuation of oxidative stress after contusion spinal cord injury through inhibition of Poly ADP Ribose Polymerase involves glutamate cysteine ligase

https://doi.org/10.1016/j.procbio.2019.05.030Get rights and content

Highlights

  • PARP1 mediated DNA repair utilizes NAD + and therefore can cause energy depletion.

  • 3-aminobenzamide inhibit Parp1 due to its structural similarity to NAD+.

  • Replenished NAD + levels regenerate glutathione (GSH) from its oxidized form (GSSH).

  • Together these changes increase GSH synthesis through upregulating Gclc.

  • These may the mechanism behind PARP1inhibition mediated beneficial effects.

Abstract

Oxidative stress-induced DNA damage in cells activates Poly ADP Ribose Polymerase 1 (PARP1) as a part of the repair process. Since it is an energy-dependent process, overactivation of PARP1 results in energy depletion and as a consequence causes parthanatos – a subtype of necrosis. Beneficial effects of inhibiting PARP1 using chemicals such as 3-aminobenzamide (3-AB) has been well documented and were shown to mitigate oxidative stress and increase anti-oxidants levels in the cells. However, the exact molecular mechanism responsible for the increased anti-oxidants levels during inhibition of PARP1 overactivation has not been established. Under these circumstances, PARP1 inhibition using 3-aminobenzamide after a contusion spinal cord injury in rats was found to increase the levels of both glutamate–cysteine ligase catalytic unit (GCLC) and glutathione (GSH). GCLC being the rate-limiting enzyme in the synthesis of GSH, the increase of GSH levels might be because of increased synthesis as well as regeneration of GSH from its oxidized state (GSSH); both the processes favored by the energy replenishment. Given that GSH is pivotal in the antioxidant defense mechanism, the observations of increased expression of Gclc as reported here might be the plausible molecular mechanism responsible for the increased anti-oxidants levels in PARP1 inhibition by 3-AB.

Introduction

Inflammation, ionizing radiation, trauma, etc. results in the cellular release of free radicals/reactive oxygen species. These entities, by their ability to donate an electron, cause oxidation of several molecules which are vital for the survival of cells [1]. This type of “oxidative stress” is considered the key reason for the delayed cell death in the penumbra of the lesion where the cells are subjected to the sub-lethal threshold of insult [2]. Under the continuum of distinct cell death sub-routines [3], cells undergo either apoptosis or necrosis based on the severity of the oxidative stress [4].

Oxidative stress in the cells can cause DNA damage and DNA-repair failure would naturally push the cells towards apoptosis. DNA damage could activate Poly ADP Ribose Polymerase 1 (PARP1) which utilizes NAD + for the repair process. When DNA damage exceeds a threshold, correspondingly, PARP1 gets overactivated leading to depletion of NAD + levels in the cells [5]. As NAD + is an indispensable co-factor required for energy metabolism (glycolysis), its depletion results in low levels of cellular ATP [6]. The consequent necrotic death of cells triggered by frank energy failure affects adjacent cells due to the spillage of toxic contents from the dying cells. A wave of delayed cell death would be triggered leading to the exacerbation of the damage caused by primary injury [7]. Thus, the futile attempt by (over activated) PARP1 proves to be fatal rather than beneficial.

Several inhibitors of PARP1 enzyme were known to block the action of forming PAR (Poly-ADP Ribose) polymer on nuclear proteins, the process that requires ATP and NAD+ [8]. Given that intracellular ATP levels serves as a molecular switch between apoptosis and necrosis [9], preventing energy depletion by PARP1 inhibition was envisaged as a way to prevent necrosis in the injured cells. Among various compounds, 3-Amninobenzamide (3-AB) was found to be a hydroxyl ion scavenger and a potent inhibitor of PARP1 [10]. It has been demonstrated in an in vitro necrotic model of PC12 cells, inhibition of over activated PARP1 using 3-AB was restoring the ATP pool thereby shifting the cell death from necrosis to apoptosis and possibly later from apoptosis to cell survival [11].

Due to the overwhelming evidence presented by experimental studies regarding the potential benefits of using PARP1 inhibitors, currently, FDA has approved the use of several PARP1 inhibitors (Olaparib, Veliparib, Niraparib, Talazoparib, Rucaparib, CEP-9722) in clinical trials targeting various cancers (for lung, prostate, breast cancers). Role of PARP1 inhibitors to mitigate oxidative stress in various neurodegenerative conditions are being vigorously investigated in the experimental studies.

Despite the voluminous literature available, the exact mechanism by which the inhibition of PARP1 resulting in increased antioxidant levels/reduced oxidative stress has not been identified.

Under these circumstances, we were interested in studying the effects of 3-AB on contusion spinal cord injury (SCI), as contusion SCI is a classic example in which PARP1 overactivation based progressive necrotic cell death sabotage the scope for recovery [12]. The inhibition of over activated PARP1 by 3-AB has been shown to prevent cell death and secondary injury [13,14]. While analyzing the various assay results, anti-oxidants levels were found to be increased as reported earlier by other authors. Interestingly, gene expressions of glutamate cysteine ligase catalytic unit (Gclc) were found to be increased significantly and resulted in increased levels of GSH and GCLC.

Glutathione is a master redox regulator that influences other enzymatic and non-enzymatic anti-oxidants [15]. Synthesis of glutathione is based on the expression of glutamate cysteine ligase which is a rate-limiting enzyme [16]. This enzyme has a catalytic unit (GCLC) and a modifier unit (GCLM); out of which the catalytic unit is involved in glutathione synthesis in the presence of ATP [17]. GCLC catalyzes the formation of gamma-glutamylcysteine from l-glutamate and cysteine. Glutathione synthetase further catalyzes the formation of glutathione (GSH) from gamma-glutamylcysteine and glycine. All these reactions require ATP.

Thus based on the observations from the present study, it is theorized that ATP replenishment and increased expression of GCLC leading to upregulation of GSH, could be the plausible mechanism by which, PARP1 inhibition results in the improved anti-oxidant defense system.

Section snippets

Materials and methods

Healthy male Wistar albino rats of 90 days old with an average weight of 200–250 g were used throughout the study. Animals were maintained in standard conditions of temperature control with unrestricted access to standard food and water under 12 h-light and dark cycle. The study design and procedures involving animals were approved by the Institutional Animal Ethics Committee, University of Madras, Taramani Campus functioning under the control of Committee for the Purpose of Control and

Effects of Parp1 inhibition on the oxidative stress caused by SCI

In the DCFH-DA assay, increased DCF fluorescence was noted in the SCI group which is indicative of increased reactive oxygen species (ROS). Similarly, lipid peroxidation levels were also increased significantly by SCI. Inhibition of PARP1 overactivation in SCI animals was found to significantly reverse these effects as shown in Fig. 1A and B.

Status of enzymatic and non-enzymatic antioxidants

The levels of enzymatic antioxidants when compared with sham operated group were significantly reduced after SCI. Inhibition of PARP1 overactivation

Discussion

In an earlier in vitro study, we had reported the hall mark features of necrosis observed in cultured neuronal cells (Neuro-2A) subjected to oxidative stress. PARP1 inhibition using 3-AB was found to marginally increase the Caspase 3 mediated apoptosis; however resulted in overall better survival of the cells [31]. This supports the possible shift of cell death from necrosis to apoptosis and later to cell survival as reported by others [11] owing to the conservation of ATP from getting

Acknowledgments

Dr. Vijaya Prakash Krishnan Muthaiah received senior research fellowship from Lady Tata Memorial Trust, Mumbai, India.

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  • Cited by (0)

    1

    Present Addresses: Department of Rehabilitation Science, School of Public Health and Health Professions, University at Buffalo, 633 Kimball Tower, Buffalo, NY 14214, USA.

    2

    Present Addresses: Department of Anatomy, Sathyabama Institute of Science and Technology Dental College and Hospital, Chennai 600119, India.

    3

    Present Addresses: Department of Electronics and Communication Engineering, Anna University, Chennai 600020, India.

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