Identification of poly‐ADP‐ribosylated mitochondrial proteins after traumatic brain injury

Abstract

Poly-ADP-ribosylation is a post-translational modification performed by poly(ADP-ribose) polymerases (PARP), involved in many diverse cellular functions including DNA repair, transcription, and long-term potentiation. Paradoxically, PARP over-activation under pathologic conditions including traumatic brain injury (TBI) results in cell death. We previously demonstrated that intra-mitochondrial poly-ADP-ribosylation occurs following excitotoxic and oxidative injury in vitro. Here we sought to identify mitochondrial proteins modified by poly-ADP-ribosylation after TBI in vivo. Poly-ADP-ribosylation within mitochondria from injured brain after experimental TBI in rats was first verified using western blot and immunoelectron microscopy. Poly-ADP-ribosylated mitochondrial proteins identified using a targeted proteomic approach included voltage-dependent anion channel-1, mitofilin, mitochondrial stress proteins, and the electron transport chain components F 1 F 0 ATPase, cytochrome c oxidase, and cytochrome c reductase. To examine the functional consequences of mitochondrial poly-ADP-ribosylation, isolated rat brain mitochondria were exposed to conditions of nitrosative stress known to activate PARP. PARP activation-induced reductions in State 3 respiration were prevented by the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone or exogenous poly(ADPribose) glycohydrolase. As the effects of PARP activation on mitochondrial respiration appear regulated by poly(ADPribose) glycohydrolase, a direct effect of poly-ADP-ribosylation on electron transport chain function is suggested. These findings may be of relevance to TBI and other diseases where mitochondrial dysfunction occurs.

Quantification of Poly(ADP-Ribose)-Modified Proteins in Cerebrospinal Fluid from Infants and Children after Traumatic Brain Injury

“…Using this assay, we were able to show an increase in CSF PAR-modified proteins and associations between male sex and increasing patient age. Increased PARP activation has been shown in multiple experimental models of TBI, including controlled cortical impact in mice and rats, and fluid percussion injury in rats (LaPlaca et al, 1998;Satchell et al, 2003;Lai et al, 2008). Importantly, a detrimental role for PARP overactivation after TBI has been shown using both PARP-1 knockout mice (B) Relationship between mean and peak CSF PAR-modified protein levels and age in TBI patients and controls.…”

“…However, during periods of severe cellular stress and energy imbalance, such as after traumatic brain injury (TBI) or stroke, overactivation of PARP can result in depletion of NAD + , energy failure, and cell death and dysfunction (Eliasson et al, 1997;Endres et al, 1997;Whalen et al, 1999;Satchell et al, 2003;Clark et al, 2007). In addition to consumption of NAD + , PARP activation can also directly inhibit electron transport, thereby reducing ATP production and energy repletion, compounding energy failure (Halmosi et al, 2001;Ha et al, 2002;Lai et al, 2008). Poly(ADP-ribose) polymerase can also mediate apoptotic cell death through the release of apoptosis-inducing factor from mitochondria (Yu et al, 2002;Du et al, 2003), a process that occurs after TBI (Zhang et al, 2002).…”

Poly(ADP-ribose) polymerases as modulators of mitochondrial activity

“…There is consensus in the literature that mitochondria possess glycohydrolase activity (the activity necessary for PAR degradation) with ARH3 being the major component [28]. Although the existence of mitochondrial PARP activity is debated, there are data that show the presence of PARylated proteins in isolated rat liver mitochondria [29,30], mitochondrial PARP activity [29,31], or identify mitochondrial malate dehydrogenase as a PARP-1 interacting protein [32]. However, these studies were based on the extraction of PARylated proteins using a PAR antibody and given the existing discrepancies between proteins identified by the different studies [29,30]; additional, non-immunological methods are required to verify these findings.…”

“…Although the existence of mitochondrial PARP activity is debated, there are data that show the presence of PARylated proteins in isolated rat liver mitochondria [29,30], mitochondrial PARP activity [29,31], or identify mitochondrial malate dehydrogenase as a PARP-1 interacting protein [32]. However, these studies were based on the extraction of PARylated proteins using a PAR antibody and given the existing discrepancies between proteins identified by the different studies [29,30]; additional, non-immunological methods are required to verify these findings. Importantly, when PARP-1 was overexpressed in mitochondria, in vitro, mitochondrial PARylation increased, which was accompanied by decreased mitochondrial output and unaltered glycolytic flux [33], highlighting a possible functional consequence of mitochondrial PARylation.…”

Neuroprotection through Stimulation of Mitochondrial Antioxidant Protein Expression

“…Metabolic depression and increased ROS production by the ETC also occurs in response to mitochondrial DNA oxidation, which is associated with neurodegenerative disorders and normal aging [11]. Finally, ROS/RNS are known to stimulate the activity of poly-ADP ribose polymerase 1 (PARP1) and the expression of P53, which in turn can cause release of mitochondrial apoptotic proteins and poly-ADP ribosylation of mitochondrial proteins [12][13][14][15].…”