PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca2 +-permeable AMPA receptors
Graphical abstract
Introduction
Poly(ADP-ribose) polymerases (PARPs) are a group of enzymes able to catalyze the transfer of ADP-ribose units from NAD+ to substrate proteins (D'Amours et al., 1999, Ame et al., 2004). PARP-1, the oldest and most studied member of the PARP group, is located in cell nuclei where it plays key roles in repairing DNA damage and controlling cell cycle and gene expression (Smith, 2001). Genotoxic agents strongly activate PARP-1 and cause an increase of PAR (the product of PARP activity) which has been shown to facilitate DNA repair and cell survival (D'Amours et al., 1999). It is therefore assumed that inhibition or knockout of PARP-1 may result in accumulation of DNA damage and eventually in cell death. This concept led to the development of PARP inhibitors for cancer treatment (Curtin and Szabo, 2013). Since the early eighties, however, Berger et al. reported that excessive activation of PARP-1 may cause cellular NAD+ and ATP depletion and a “metabolic catastrophe” or “PARP-induced suicide” which was considered an “evolutionary strategy” of multicellular organisms to prevent the survival of excessively damaged cells (Berger, 1985). More recently, it was reported that PARP is involved in glutamate NMDA receptor-dependent neuronal death (Cosi et al., 1994). The NMDA-mediated Ca2 + inflow activates neuronal nitric oxide synthase (nNOS) and a number of other enzymes (Pieper et al., 1999) leading to the production of free radical species that cause DNA strand breaks (Giovannelli et al., 2002, Goto et al., 2002), PARP activation, NAD+/ATP depletion and neuronal loss (Dawson et al., 1991, Zhang et al., 1994). In the last few years a wealth of data linking excessive PARP activation to cell demise emerged (Virag et al., 2013). In particular, PARP activation may cause mitochondrial AIF release, AIF translocation to the nucleus, chromatin disruption and a caspase-independent cell death (parthanatos) (Andrabi et al., 2006, Yu et al., 2006). PARP-1 activation has been shown to cause cell death by triggering a signaling cascade involving the MAP kinase JNK (Xu et al., 2006). Intracellular PAR polymer accumulation and the resulting increase of ADP ribose has been shown to open the transient receptor potential 2 (TRPM2) channels and allow the entry of an excessive amount of Ca2 + in excitable cells (Blenn et al., 2011). Other proposed PARP-mediated cell death mechanisms involve the metabolic sensing enzymes AMPK and mTOR (Ethier et al., 2012). Studies aimed at clarifying the mechanisms of PARP-induced cell death in brain could be particularly important because an excessive activation of the enzyme seems to contribute to neuronal death in stroke, brain trauma, Parkinson and other degenerative processes (Virag and Szabo, 2002, Moroni, 2008, Besson, 2009, Xu et al., 2014, Lee et al., 2013).
In the present study, we exposed organotypic hippocampal slices to N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG), a commonly used PARP-activating agent (Kupper et al., 1995, Ha and Snyder, 1999). We noticed that MNNG causes a rather peculiar and selective cellular toxicity affecting the principal neurons of the CA1 hippocampal region. The anatomical pattern of neuronal loss was comparable to that induced, in the same preparation, by oxygen and glucose deprivation (OGD) (Pellegrini-Giampietro et al., 1999b, Meli et al., 2004, Scartabelli et al., 2008, Moroni et al., 2009). We investigated the mechanisms underlying this selective toxicity with biochemical, morphological and electrophysiological techniques and found that PARP-1 activation was associated with increased expression of Ca2 +-permeable AMPA receptors in CA1 but not CA3 pyramidal neurons.
Section snippets
Materials and methods
Experiments and animal use procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). The experimental protocols were approved by the Animal Care Committee of the Department of Pharmacology, University of Florence, in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123) and the European Communities
MNNG induces selective and delayed CA1 pyramidal cell death in rat organotypic hippocampal slices
To evaluate the role of PARP activation on neuronal survival, we set up an in vitro model of toxicity by exposing organotypic hippocampal slices to different concentrations (0.1–1 mM) of MNNG for various periods of time. MNNG induced a dose-dependent neurodegeneration in hippocampal slices. When MNNG was incubated at 100 μM for 5 min, we observed a selective degeneration of the CA1 subfield. CA1 damage was delayed, in that it could not be observed until 48 h after the exposure to MNNG (Figs. 1A and
Discussion
The mechanisms of cell protection or cell demise promoted by PARP activation in the CNS have been widely studied in the recent past and a number of interesting processes leading either to cell survival or to cell loss after differential activation of the members of the PARP family have been identified (Moroni et al., 2009, Gerace et al., 2012, Virag et al., 2013). Most of the available studies have been carried out in cell lines (Ha and Snyder, 1999, Fossati et al., 2006, Ethier et al., 2012),
Conflict of interest
The authors declare no conflict of interest. Dr. Flavio Moroni has patent and patent applications on PARP inhibitors.
Acknowledgments
This work was supported by the University of Florence; and Ente Cassa di Risparmio di Firenze Grants to F.M, G.M and to T.M.
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